Cleavable linker compositions and methods

ABSTRACT

Provided herein are cleavable linkers, pharmaceutical compositions thereof, as well as nucleic acids, and methods for making and discovering the same. The cleavable linkers described herein have improved efficacy and safety.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/398,500, filed on Aug. 10, 2021, which claims the benefit of U.S.Provisional Application No. 63/064,268, filed Aug. 11, 2020, whichapplication is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Oct. 18, 2022, isnamed 52426-720_301 SL.xml and is 42,044 bytes in size.

SUMMARY

Disclosed herein, in certain embodiments, are isolated polypeptidescomprising a cleavable linker according to the amino acid sequence ofSEQ ID NO: 1 (LSGRSDAG). In some embodiments, the cleavable linkercomprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). Insome embodiments, the cleavable linker comprises the amino acid sequenceof SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the cleavablelinker comprises the amino acid sequence of SEQ ID NO: 4(AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linkercomprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). Insome embodiments, the cleavable linker comprises the amino acid sequenceof SEQ ID NO: 6 (LSGRSDAGSPLGLAG). In some embodiments, the cleavablelinker is cleavable by a protease. In some embodiments, the proteasecomprises a tumor specific protease. In some embodiments, the proteasecomprises a matrix metalloprotease (MMP) or a serine protease. In someembodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9,MMP13, or MMP14. In some embodiments, the serine protease comprisesmatriptase, urokinase, or hepsin. In some embodiments, the isolatedpolypeptide further comprises an antigen binding domain that binds to atarget antigen. In some embodiments, the antigen binding domain isC-terminal to the cleavable linker. In some embodiments, the isolatedpolypeptide further comprises a cytokine or cytokine fragment that bindsto a cytokine receptor. In some embodiments, the cytokine or cytokinefragment is C-terminal to the cleavable linker. In some embodiments, thecleavable linker connects a peptide to an antigen binding domain thatbinds to a target antigen or to a cytokine or cytokine fragment thatbinds to a cytokine receptor in a configuration according to Formula I:A₁-L₁-P₁ wherein A₁ comprises the antigen binding domain that binds tothe target antigen or the cytokine or cytokine fragment that binds tothe cytokine receptor; L₁ comprises the cleavable linker; P₁ comprises apeptide that impairs binding of the antigen binding domain to the targetantigen or impairs binding of the cytokine to the cytokine receptor. Insome embodiments, P₁ is connected N-terminal to the cleavable linker andA₁ is connected C-terminal to the cleavable linker. In some embodiments,P₁ is connected C-terminal to the cleavable linker and A₁ is connectedN-terminal to the cleavable linker. In some embodiments, P₁ is bound toA₁ through ionic interactions, electrostatic interactions, hydrophobicinteractions, Pi-stacking interactions, and H-bonding interactions, or acombination thereof. In some embodiments, P₁ has less than 70% sequencehomology to the target antigen or the cytokine receptor. In someembodiments, P₁ comprises a peptide sequence of at least 10 amino acidsin length. In some embodiments, P₁ comprises a peptide sequence of atleast 10 amino acids in length and no more than 20 amino acids inlength. In some embodiments, P₁ comprises a peptide sequence of at least16 amino acids in length. In some embodiments, P₁ comprises a peptidesequence of no more than 40 amino acids in length. In some embodiments,P₁ comprises a cyclic peptide or a linear peptide. In some embodiments,P₁ comprises a cyclic peptide. In some embodiments, P₁ is further linkedto a half-life extending moiety. In some embodiments, the half-lifeextending moiety is a single-domain antibody. In some embodiments, thesingle domain antibody comprises 10G. In some embodiments, A₁ comprisesan antibody, a single chain variable fragment (scFv), a heavy chainvariable domain (VH domain), a light chain variable domain (VL domain),a variable domain (VHH) of a camelid derived single domain antibody, aFab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chainpolypeptide. In some embodiments, the target antigen comprises a tumorantigen. In some embodiments, A₁ comprises the Fab light chainpolypeptide or the Fab heavy chain polypeptide. In some embodiments, A₁comprises an epidermal growth factor receptor (EGFR) binding domain. Insome embodiments, the target antigen comprises an effector cell antigen.In some embodiments, A₁ comprises the scFv. In some embodiments, thescFv comprises an anti-CD3e single chain variable fragment. In someembodiments, A₁ comprises the cytokine. In some embodiments, thecytokine or cytokine fragment is a wild-type cytokine. In someembodiments, the cytokine or cytokine fragment is a mutein of thecytokine. In some embodiments, the cytokine receptor is an interferonreceptor or an interleukin receptor. In some embodiments, the cytokinereceptor comprises an interferon receptor, GM-CSF receptor, IL-2receptor, IL-4 receptor, IL-6 receptor, IL-7 receptor, IL-10 receptor,IL-12 receptor, IL-15 receptor, IL-21 receptor, or TGF-β receptor. Insome embodiments, the cytokine or cytokine fragment comprises aninterferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In someembodiments, the cytokine or cytokine fragment comprises an IL-2, IL-12,IL-6, IL-4, IL-10, or TGFβ. In some embodiments, the isolatedpolypeptide is complexed with a second isolated polypeptide comprising asecond antigen binding domain or a second cytokine or second cytokinefragment. In some embodiments, the second isolated polypeptide is in aconfiguration according to Formula II: A₂-L₂-P₂ wherein A₂ comprises thesecond antigen binding domain or the second cytokine; L₂ comprises asecond cleavable linker; P₂ comprises a second peptide that impairsbinding of the second antigen binding domain to a second target antigenor impairs binding of the second cytokine or second cytokine fragment toa second cytokine receptor. In some embodiments, the second cleavablelinker comprises the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). Insome embodiments, the second cleavable linker comprises the amino acidsequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, thesecond cleavable linker comprises the amino acid sequence of SEQ ID NO:26 (AGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linkercomprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).In some embodiments, the second cleavable linker comprises the aminoacid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, thesecond cleavable linker comprises the amino acid sequence of SEQ ID NO:6 (LSGRSDAGSPLGLAG). In some embodiments, P₂ is connected N-terminal tothe second cleavable linker and A₂ is connected C-terminal to the secondcleavable linker. In some embodiments, P₂ is connected C-terminal to thesecond cleavable linker and A₂ is connected N-terminal to the secondcleavable linker. In some embodiments, P₂ is bound to A₂ through ionicinteractions, electrostatic interactions, hydrophobic interactions,Pi-stacking interactions, and H-bonding interactions, or a combinationthereof. In some embodiments, P₂ has less than 70% sequence homology tothe second target antigen or the second cytokine receptor. In someembodiments, P₂ comprises a peptide sequence of at least 10 amino acidsin length. In some embodiments, P₂ comprises a peptide sequence of atleast 10 amino acids in length and no more than 20 amino acids inlength. In some embodiments, P₂ comprises a peptide sequence of at least16 amino acids in length. In some embodiments, P₂ comprises a peptidesequence of no more than 40 amino acids in length. In some embodiments,P₂ comprises a cyclic peptide or a linear peptide. In some embodiments,P₂ comprises a cyclic peptide. In some embodiments, A₂ comprises anantibody, a single chain variable fragment (scFv), a heavy chainvariable domain (VH domain), a light chain variable domain (VL domain),a variable domain (VHH) of a camelid derived single domain antibody, aFab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chainpolypeptide. In some embodiments, the second target antigen comprises atumor antigen. In some embodiments, A₂ comprises the Fab light chainpolypeptide or the Fab heavy chain polypeptide. In some embodiments, A₂comprises an epidermal growth factor receptor (EGFR) binding domain. Insome embodiments, the second target antigen comprises an effector cellantigen. In some embodiments, A₂ comprises the scFv. In someembodiments, the scFv comprises an anti-CD3e single chain variablefragment. In some embodiments, A₂ comprises the second cytokine. In someembodiments, the second cytokine or second cytokine fragment is awild-type cytokine. In some embodiments, the second cytokine or secondcytokine fragment is a mutein of the cytokine. In some embodiments, thesecond cytokine receptor is an interferon receptor or an interleukinreceptor. In some embodiments, the second cytokine receptor comprises aninterferon receptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor,IL-21 receptor, or TGF-β receptor. In some embodiments, the secondcytokine or second cytokine fragment comprises an interferon, GM-CSF,IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the secondcytokine or second cytokine fragment comprises an IL-2, IL-12, IL-6,IL-4, IL-10, or TGFβ.

Disclosed herein are pharmaceutical compositions comprising: theisolated polypeptide comprising a cleavable linker according to any ofthe above embodiments; and a pharmaceutically acceptable excipient.

Disclosed herein are isolated recombinant nucleic acid molecule encodingthe isolated polypeptide comprising a cleavable linker according to anyof the above embodiments.

Disclosed herein are vectors comprising the recombinant nucleic acidmolecule according to the above embodiment.

Disclosed herein are methods of producing an isolated polypeptidecomprising a cleavable linker comprising culturing a cell underconditions that lead to expression of the polypeptide, wherein the cellcomprises the vector of the above embodiment.

Disclosed herein are methods of manufacturing an isolated polypeptidecomprising a cleavable linker, the method comprising: (a) culturing acell comprising the recombinant nucleic acid molecule of the aboveembodiments under conditions that lead to expression of the polypeptide,and (b) isolating the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIGS. 1A-1B illustrate binding of polypeptide complexes PC-1, PC-2,PC-3, PC-4, and PC-5 comprising EGFR masking (FIG. 1A) and followed bycleavage by the tumor protease MTSP1 (FIG. 1B).

FIGS. 2A-2B illustrate of polypeptide complexes PC-1, PC-2, PC-3, PC-4,and PC-5 comprising CD3ε masking (FIG. 2A) and followed by cleavage bythe tumor protease MTSP1 (FIG. 2B).

FIGS. 3A-3B illustrate binding of polypeptide complexes PC-2, PC-3,PC-4, PC-5, PC-1, and PC-6 to EGFR-biotin (FIG. 3A) and CD3ε-biotin(FIG. 3B) measured by ELISA.

FIGS. 4A-4E illustrate cytotoxicity against tumor target cells HCT116for polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6.

FIGS. 5A-5D illustrate pharmacokinetics of polypeptide PC-1, PC-2, PC-3,PC-7, PC-4, and PC-5 in cynomolgus monkey.

FIGS. 6A-6D illustrate cytokine release of polypeptide complexes PC-1,PC-2, PC-3, PC-7, PC-4, and PC-5 molecules in cynomolgus monkey.

FIG. 7A-7B illustrates graphs of AST and ALT levels of polypeptidecomplexes PC-1, PC-2, PC-3, PC-4, and PC-5 in cynomolgus monkey.

DETAILED DESCRIPTION

Protein-based therapies such as antibodies, T cell receptors (TCR), andcytokine therapies have proven effective for a variety diseases anddisorders. As with any therapy, there is a need to minimize off-targeteffects of the protein-based therapy in healthy tissue while maintainingactivity of the protein-based therapy in disease tissue. One suchstrategy is to create an inactive form of the protein-based therapy inwhich a necessary binding site on the protein-based therapy is blockedwith a peptide linked to the protein-based therapy, thereby preventingthe protein-based therapy from binding or interacting with its cognatereceptor or target antigen when in healthy tissue. For activating theprotein-based therapy in the desired disease-state microenvironment, thepeptide is linked to the protein-based therapy with a linker that iscleavable by a protease that is specific to the disease-statemicroenvironment. The peptide is then released from the protein-basedtherapy when in the disease-state microenvironment.

Accordingly, disclosed herein, are cleavable linkers which can beapplied to a variety of protein-based therapy formats, for use inreducing off-target effects of the protein-based therapy in healthytissue, while maintaining activity of the protein-based therapy indisease tissue. The cleavable linkers, as disclosed herein, havedesirable properties, which include, for example but are not limited to,increased rates of proteolysis by tumor proteases or cleavable by anexpanded panel of tumor proteases while also having comparable safetyprofiles relative to control linkers.

Certain Definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the given value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” should be assumed to mean an acceptable error range for theparticular value.

“Fragment” as used herein refers to a peptide or a polypeptide thatcomprises less than the full length amino acid sequence.

“Peptide”, “P₁”, or “P₂” as used herein refers to an amino acid sequenceof less than 50 amino acids and specifically excludes a cytokine ligandbinding domain, fragments, or muteins thereof, a cytokine receptor,fragments, or muteins thereof, and any antibody or antibody bindingfragments (for example, a single domain antibody, Fab, or scFv) thatbinds to a cytokine, or binds to a cognate cytokine receptor.

As disclosed herein, in some embodiments, are isolated polypeptidescomprising a cleavable linker according to the amino acid sequence ofSEQ ID NO: 1 (LSGRSDAG)

In some embodiments, the cleavable linker comprises the amino acidsequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, thecleavable linker comprises the amino acid sequence of SEQ ID NO: 26(AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprisesthe amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In someembodiments, the cleavable linker comprises the amino acid sequence ofSEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the cleavable linkercomprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

In some embodiments, the cleavable linker consists of the amino acidsequence of SEQ ID NO: 1 (LSGRSDAG). In some embodiments, the cleavablelinker consists of the amino acid sequence of SEQ ID NO: 3(ISSGLLSGRSDAG). In some embodiments, the cleavable linker consists ofthe amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In someembodiments, the cleavable linker consists of the amino acid sequence ofSEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavablelinker consists of the amino acid sequence of SEQ ID NO: 5(SPLGLSGRSDAG). In some embodiments, the cleavable linker consists ofthe amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

In some embodiments, the cleavable linker comprises the amino acidsequence selected from the group consisting of SEQ ID NO: 3, 4, 5, and6.

In some embodiments, are isolated polypeptides comprising a cleavablelinker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG)SEQ ID NO: 3, Linker 2 (AAGLLAPPGGLSGRSDAG) SEQ ID NO:4, Linker 3(SPLGLSGRSDAG) SEQ ID NO: 5, or Linker 4 (LSGRSDAGSPLGLAG) SEQ ID NO: 6or an isolated polypeptide comprising a cleavable linker that has 1, 2,or 3 amino acid substitutions, additions, or deletions relative to theamino acid sequence of Linker 1, Linker 2, Linker 3, or Linker 4.

In some embodiments, the cleavable linker comprises the amino acidsequence of Linker 1. In some embodiments, the cleavable linker consistsof the amino acid sequence of Linker 1. In some embodiments, thecleavable linker has 1 amino acid substitution, addition, or deletionrelative to the amino acid sequence of Linker 1. In some embodiments,the cleavable linker has 2 amino acid substitutions, additions, ordeletions relative to the amino acid sequence of Linker 1. In someembodiments, the cleavable linker has 3 amino acid substitutions,additions, or deletions relative to the amino acid sequence of Linker 1.

In some embodiments, the cleavable linker comprises the amino acidsequence of Linker 2. In some embodiments, the cleavable linker consistsof the amino acid sequence of Linker 2. In some embodiments, thecleavable linker has 1 amino acid substitution, addition, or deletionrelative to the amino acid sequence of Linker 2. In some embodiments,the cleavable linker has 2 amino acid substitutions, additions, ordeletions relative to the amino acid sequence of Linker 2. In someembodiments, the cleavable linker has 3 amino acid substitutions,additions, or deletions relative to the amino acid sequence of Linker 2.

In some embodiments, the cleavable linker comprises the amino acidsequence of Linker 3. In some embodiments, the cleavable linker consistsof the amino acid sequence of Linker 3. In some embodiments, thecleavable linker has 1 amino acid substitution, addition, or deletionrelative to the amino acid sequence of Linker 3. In some embodiments,the cleavable linker has 2 amino acid substitutions, additions, ordeletions relative to the amino acid sequence of Linker 3. In someembodiments, the cleavable linker has 3 amino acid substitutions,additions, or deletions relative to the amino acid sequence of Linker 3.

In some embodiments, the cleavable linker comprises the amino acidsequence of Linker 4. In some embodiments, the cleavable linker consistsof the amino acid sequence of Linker 4. In some embodiments, thecleavable linker has 1 amino acid substitution, addition, or deletionrelative to the amino acid sequence of Linker 4. In some embodiments,the cleavable linker has 2 amino acid substitutions, additions, ordeletions relative to the amino acid sequence of Linker 4. In someembodiments, the cleavable linker has 3 amino acid substitutions,additions, or deletions relative to the amino acid sequence of Linker 4.

In some embodiments, the amino acid substitution, addition, or deletionresults in an amino acid sequence that is at least 75% identical, e.g.,77%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the amino acid sequence of any proteindescribed herein. In some embodiments, the amino acid substitution is aconservative amino acid substitution. Among the common amino acids, forexample, a “conservative amino acid substitution” is illustrated by asubstitution among amino acids within each of the following groups: (1)glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine,tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate andglutamate, (5) glutamine and asparagine, and (6) lysine, arginine andhistidine.

In some embodiments, the cleavable linker comprises a modified aminoacid or non-natural amino acid, or a modified non-natural amino acid, ora combination thereof. In some embodiments, the modified amino acid or amodified non-natural amino acid comprises a post-translationalmodification. In some embodiments, the cleavable linker comprises amodification including, but not limited, to acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Modifications aremade anywhere to the cleavable linker including the peptide backbone, orthe amino acid side chains.

In some embodiments, the cleavable linker is cleavable by a protease. Insome embodiments, the protease is present in higher levels in adisease-state microenvironment relative to levels in healthy tissue or amicroenvironment that is not the disease-state microenvironment. In someembodiments, the protease comprises a tumor specific protease. In someembodiments, the protease comprises a matrix metalloprotease (MMP) or aserine protease. In some embodiments, the matrix metalloproteasecomprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, thematrix metalloprotease is selected from the group consisting of MMP2,MMP7, MMP9, MMP13, and MMP14. In some embodiments, the matrixmetalloprotease comprises MMP2. In some embodiments, the matrixmetalloprotease comprises MMP7. In some embodiments, the matrixmetalloprotease comprises MMP9. In some embodiments, the matrixmetalloprotease comprises MMP13. In some embodiments, the matrixmetalloprotease comprises MMP14. In some embodiments, the serineprotease comprises matriptase, urokinase, or hepsin. In someembodiments, the serine protease is selected from the group consistingof matriptase, urokinase, and hepsin. In some embodiments, the serineprotease comprises matriptase. In some embodiments, the serine proteasecomprises urokinase. In some embodiments, the serine protease compriseshepsin. In some embodiments, the cleavable linker is cleaved by avariety of proteases. In some embodiments, the cleavable linker iscleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than20 different proteases.

In some embodiments, the cleavable linker has increased rates ofproteolysis as compared to the rates of proteolysis for linkers withoutthe cleavable linker sequences. In some embodiments, the cleavablelinker has increased rates of proteolysis that is at least 5× higherthan the rates of proteolysis for linkers without the cleavable linkersequences. In some embodiments, the cleavable linker has increased ratesof proteolysis that is at least 8× higher than the rates of proteolysisfor linkers without the cleavable linker sequences. In some embodiments,the cleavable linker has increased rates of proteolysis that is at least10× higher than the rates of proteolysis for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas increased rates of proteolysis that is at least 15× higher than therates of proteolysis for linkers without the cleavable linker sequences.In some embodiments, the cleavable linker has increased rates ofproteolysis that is at least 20× higher than the rates of proteolysisfor linkers without the cleavable linker sequences. In some embodiments,the cleavable linker has increased rates of proteolysis that is at least25× higher than the rates of proteolysis for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas increased rates of proteolysis that is at least 30× higher than therates of proteolysis for linkers without the cleavable linker sequences.In some embodiments, the cleavable linker has increased rates ofproteolysis that is at least 40× higher than the rates of proteolysisfor linkers without the cleavable linker sequences. In some embodiments,the cleavable linker has increased rates of proteolysis that is at least50× higher than the rates of proteolysis for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas increased rates of proteolysis that is at least 60× higher than therates of proteolysis for linkers without the cleavable linker sequences.In some embodiments, the cleavable linker has increased rates ofproteolysis that is at least 70× higher than the rates of proteolysisfor linkers without the cleavable linker sequences. In some embodiments,the cleavable linker has increased rates of proteolysis that is at least75× higher than the rates of proteolysis for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas increased rates of proteolysis that is at least 80× higher than therates of proteolysis for linkers without the cleavable linker sequences.In some embodiments, the cleavable linker has increased rates ofproteolysis that is at least 90× higher than the rates of proteolysisfor linkers without the cleavable linker sequences. In some embodiments,the cleavable linker has increased rates of proteolysis that is at least100× higher than the rates of proteolysis for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas increased rates of proteolysis that is at least 120× higher than therates of proteolysis for linkers without the cleavable linker sequences.In some embodiments, the cleavable linker is cleaved by a protease. Insome embodiments, the protease comprises a tumor specific protease. Insome embodiments, the protease comprises a matrix metalloprotease (MMP)or a serine protease. In some embodiments, the matrix metalloproteasecomprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, theserine protease comprises matriptase, urokinase, or hepsin.

In some embodiments, the cleavable linker has improved stability inhuman serum as compared to the stability in human serum without thecleavable linker sequences. In some embodiments, the cleavable linkerhas improved stability in human serum that is at least 5× higher thanthe stability in human serum for linkers without the cleavable linkersequences. In some embodiments, the cleavable linker has improvedstability in human serum that is at least 8× higher than the stabilityin human serum for linkers without the cleavable linker sequences. Insome embodiments, the cleavable linker has improved stability in humanserum that is at least 10× higher than the stability in human serum forlinkers without the cleavable linker sequences. In some embodiments, thecleavable linker has improved stability in human serum that is at least15× higher than the stability in human serum for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas improved stability in human serum that is at least 20× higher thanthe stability in human serum for linkers without the cleavable linkersequences. In some embodiments, the cleavable linker has improvedstability in human serum that is at least 25× higher than the stabilityin human serum for linkers without the cleavable linker sequences. Insome embodiments, the cleavable linker has improved stability in humanserum that is at least 30× higher than the stability in human serum forlinkers without the cleavable linker sequences. In some embodiments, thecleavable linker has improved stability in human serum that is at least40× higher than the stability in human serum for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas improved stability in human serum that is at least 50× higher thanthe stability in human serum for linkers without the cleavable linkersequences. In some embodiments, the cleavable linker has improvedstability in human serum that is at least 60× higher than the stabilityin human serum for linkers without the cleavable linker sequences. Insome embodiments, the cleavable linker has improved stability in humanserum that is at least 70× higher than the stability in human serum forlinkers without the cleavable linker sequences. In some embodiments, thecleavable linker has improved stability in human serum that is at least75× higher than the stability in human serum for linkers without thecleavable linker sequences. In some embodiments, the cleavable linkerhas improved stability in human serum that is at least 80× higher thanthe stability in human serum for linkers without the cleavable linkersequences. In some embodiments, the cleavable linker has improvedstability in human serum that is at least 90× higher than the stabilityin human serum for linkers without the cleavable linker sequences. Insome embodiments, the cleavable linker has improved stability in humanserum that is at least 100× higher than the stability in human serum forlinkers without the cleavable linker sequences. In some embodiments, thecleavable linker has improved stability in human serum that is at least120× higher than the stability in human serum for linkers without thecleavable linker sequences.

In some embodiments, the isolated polypeptide comprising the cleavablelinker has increased rates of proteolysis as compared to an isolatedpolypeptide of the same amino acid sequence but comprising a cleavablelinker according to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the isolated polypeptide comprising the cleavablelinker has improved or equivalent serum stability as compared to anisolated polypeptide of the same amino acid sequence but comprising acleavable linker according to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the isolated polypeptide comprising the cleavablelinker has improved or equivalent in vitro tumor cell killing ascompared to an isolated polypeptide of the same amino acid sequence butcomprising a cleavable linker according to the amino acid sequence ofSEQ ID NO: 2.

In some embodiments, the isolated polypeptide comprising the cleavablelinker has improved or equivalent pharmacokinetic parameters incynomolgus monkeys as compared to an isolated polypeptide of the sameamino acid sequence but comprising a cleavable linker according to theamino acid sequence of SEQ ID NO: 2.

In some embodiments, the isolated polypeptide comprising the cleavablelinker has improved or equivalent liver toxicity levels in cynomolgusmonkeys as compared to an isolated polypeptide of the same amino acidsequence but comprising a cleavable linker according to the amino acidsequence of SEQ ID NO: 2.

In some embodiments, the isolated polypeptide further comprises anantigen binding domain that binds to a target antigen. In someembodiments, the antigen binding domain is C-terminal to the cleavablelinker. In some embodiments, the isolated polypeptide further comprisesa cytokine or cytokine fragment that binds to a cytokine receptor. Insome embodiments, the cytokine or cytokine fragment is C-terminal to thecleavable linker.

In some embodiments, the cleavable linker connects a peptide to anantigen binding domain that binds to a target antigen or to a cytokinethat binds to a cytokine receptor in a configuration according toFormula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ comprises the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁comprises the cleavable linker; P₁ comprises a peptide that impairsbinding of the antigen binding domain to the target antigen or impairsbinding of the cytokine to the cytokine receptor. In some embodiments,P₁ is connected N-terminal to the cleavable linker and A₁ is connectedC-terminal to the cleavable linker. In some embodiments, P₁ is connectedC-terminal to the cleavable linker and A₁ is connected N-terminal to thecleavable linker.

In some embodiments, the isolated polypeptide is complexed with a secondisolated polypeptide comprising a second antigen binding domain or asecond cytokine. In some embodiments, the second isolated polypeptide isin a configuration according to Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ comprises the second antigen binding domain or the secondcytokine; L₂ comprises a second cleavable linker; and P₂ comprises asecond peptide that impairs binding of the second antigen binding domainto a second target antigen or impairs binding of the second cytokine toa second cytokine receptor.

In some embodiments, P₂ is connected N-terminal to the second cleavablelinker and A₂ is connected C-terminal to the second cleavable linker. Insome embodiments, P₂ is connected C-terminal to the second cleavablelinker and A₂ is connected N-terminal to the second cleavable linker.

In some embodiments, the second cleavable linker comprises the aminoacid sequence of SEQ ID NO: 1 (LSGRSDAG). In some embodiments, thesecond cleavable linker comprises the amino acid sequence of SEQ ID NO:3 (ISSGLLSGRSDAG). In some embodiments, the second cleavable linkercomprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).In some embodiments, the second cleavable linker comprises the aminoacid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments,the second cleavable linker comprises the amino acid sequence of SEQ IDNO: 5 (SPLGLSGRSDAG). In some embodiments, the second cleavable linkercomprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

In some embodiments, L₁ or L₂ is at least 8 amino acids in length. Insome embodiments, L₁ or L₂ is at least 10 amino acids in length but nomore than 50 amino acids in length. In some embodiments, L₁ or L₂ is atleast 10 amino acids in length but no more than 30 amino acids inlength. In some embodiments, L₁ or L₂ is at least 18 amino acids inlength. In some embodiments, L₁ or L₂ is at least 26 amino acids inlength. In some embodiments, L₁ or L₂ is at least 30 amino acids inlength. In some embodiments, L₁ or L₂ is at least 40 amino acids inlength. In some embodiments, L₁ or L₂ is at least 50 amino acids inlength.

Peptide (P₁ or P₂)

In some embodiments, P₁ comprises a peptide that impairs binding of theantigen binding domain to the target antigen. In some embodiments, P₁comprises a peptide that impairs binding of the cytokine to the cytokinereceptor. In some embodiments, P₁ is bound to A₁ through ionicinteractions, electrostatic interactions, hydrophobic interactions,Pi-stacking interactions, and H-bonding interactions, or a combinationthereof. In some embodiments, P₁ is bound to A₁ at or near a cytokinereceptor binding site. In some embodiments, P₁ is bound to A₁ at or nearan antigen binding site. In some embodiments, P₁ becomes unbound from A₁when L₁ is cleaved by the protease thereby exposing P₁ to the targetantigen or cytokine receptor. In some embodiments, the proteasecomprises a tumor specific protease. In some embodiments, the proteasecomprises a matrix metalloprotease (MMP) or a serine protease. In someembodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9,MMP13, or MMP14. In some embodiments, the serine protease comprisesmatriptase, urokinase, or hepsin. In some embodiments, P₁ impairsbinding of A₁ to the target antigen or cytokine receptor by non-stericblocking. In some embodiments, P₁ impairs binding of A₁ to the targetantigen or cytokine receptor through covalent interactions. In someembodiments, P₁ is not a cytokine, cytokine binding fragment, cytokinemutein, or combinations thereof of the cognate receptor of the cytokine.In some embodiments, A₁ is not an antibody or fragment thereof thatbinds to the cytokine receptor.

In some embodiments, P₂ comprises a peptide that impairs binding of thesecond antigen binding domain to the second target antigen. In someembodiments, P₂ comprises a peptide that impairs binding of the secondcytokine to the second cytokine receptor. In some embodiments, P₂ isbound to A₂ through ionic interactions, electrostatic interactions,hydrophobic interactions, Pi-stacking interactions, and H-bondinginteractions, or a combination thereof. In some embodiments, P₂ is boundto A₂ at or near a cytokine receptor binding site. In some embodiments,P₂ is bound to A₂ at or near an antigen binding site. In someembodiments, P₂ becomes unbound from A₂ when L₂ is cleaved by theprotease thereby exposing P₂ to the second target antigen or secondcytokine receptor. In some embodiments, the protease comprises a tumorspecific protease.

In some embodiments, the protease comprises a matrix metalloprotease(MMP) or a serine protease. In some embodiments, the matrixmetalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In someembodiments, the serine protease comprises matriptase, urokinase, orhepsin. In some embodiments, P₂ impairs binding of A₂ to the secondtarget antigen or second cytokine receptor by non-steric blocking. Insome embodiments, P₂ impairs binding of A₂ to the second target antigenor second cytokine receptor through covalent interactions. In someembodiments, P₂ is not a cytokine, cytokine binding fragment, cytokinemutein, or combinations thereof of the cognate receptor of the cytokine.In some embodiments, A₂ is not an antibody or fragment thereof thatbinds to the cytokine receptor.

In some embodiments, P₁ has less than 70% sequence homology to thetarget antigen. In some embodiments, P₁ has less than 75% sequencehomology to the target antigen. In some embodiments, P₁ has less than80% sequence homology to the target antigen. In some embodiments, P₁ hasless than 85% sequence homology to the target antigen. In someembodiments, P₁ has less than 90% sequence homology to the targetantigen. In some embodiments, P₁ has less than 95% sequence homology tothe target antigen. In some embodiments, P₁ has less than 98% sequencehomology to the target antigen. In some embodiments, P₁ has less than99% sequence homology to the target antigen.

In some embodiments, P₁ has less than 70% sequence homology to thecytokine receptor. In some embodiments, P₁ has less than 75% sequencehomology to the cytokine receptor. In some embodiments, P₁ has less than80% sequence homology to the cytokine receptor. In some embodiments, P₁has less than 85% sequence homology to the cytokine receptor. In someembodiments, P₁ has less than 90% sequence homology to the cytokinereceptor. In some embodiments, P₁ has less than 95% sequence homology tothe cytokine receptor. In some embodiments, P₁ has less than 98%sequence homology to the cytokine receptor. In some embodiments, P₁ hasless than 99% sequence homology to the cytokine receptor.

In some embodiments, P₂ has less than 70% sequence homology to thesecond target antigen. In some embodiments, P₂ has less than 75%sequence homology to the second target antigen. In some embodiments, P₂has less than 80% sequence homology to the second target antigen. Insome embodiments, P₂ has less than 85% sequence homology to the secondtarget antigen. In some embodiments, P₂ has less than 90% sequencehomology to the second target antigen. In some embodiments, P₂ has lessthan 95% sequence homology to the second target antigen. In someembodiments, P₂ has less than 98% sequence homology to the second targetantigen. In some embodiments, P₂ has less than 99% sequence homology tothe second target antigen.

In some embodiments, P₂ has less than 70% sequence homology to thesecond cytokine receptor. In some embodiments, P₂ has less than 75%sequence homology to the second cytokine receptor. In some embodiments,P₂ has less than 80% sequence homology to the second cytokine receptor.In some embodiments, P₂ has less than 85% sequence homology to thesecond cytokine receptor. In some embodiments, P₂ has less than 90%sequence homology to the second cytokine receptor. In some embodiments,P₂ has less than 95% sequence homology to the second cytokine receptor.In some embodiments, P₂ has less than 98% sequence homology to thesecond cytokine receptor. In some embodiments, P₂ has less than 99%sequence homology to the second cytokine receptor.

In some embodiments, P₁ or P₂ comprises a de novo amino acid sequencethat shares less than 50% sequence homology to a cytokine, cytokinereceptor, or antibody or fragments thereof that bind to the cytokine orcytokine receptor. In some embodiments, P₁ or P₂ comprises a de novoamino acid sequence that shares less than 40% sequence homology to acytokine, cytokine receptor, or antibody or fragments thereof that bindto the cytokine or cytokine receptor. In some embodiments, P₁ or P₂comprises a de novo amino acid sequence that shares less than 30%sequence homology to a cytokine, cytokine receptor, or antibody orfragments thereof that bind to the cytokine or cytokine receptor. Insome embodiments, P₁ or P₂ comprises a de novo amino acid sequence thatshares less than 20% sequence homology to a cytokine, cytokine receptor,or antibody or fragments thereof that bind to the cytokine or cytokinereceptor. In some embodiments, P₁ or P₂ comprises a de novo amino acidsequence that shares less than 10% sequence homology to a cytokine,cytokine receptor, or antibody or fragments thereof that bind to thecytokine or cytokine receptor. In some embodiments, P₁ or P₂ isidentified from a peptide library that contains random amino acidsequences.

In some embodiments, P₁ or P₂ comprises a de novo amino acid sequencethat shares less than 50% sequence homology to the target antigen. Insome embodiments, P₁ or P₂ comprises a de novo amino acid sequence thatshares less than 40% sequence homology to the target antigen. In someembodiments, P₁ or P₂ comprises a de novo amino acid sequence thatshares less than 30% sequence homology to the target antigen. In someembodiments, P₁ or P₂ comprises a de novo amino acid sequence thatshares less than 20% sequence homology to the target antigen. In someembodiments, P₁ or P₂ comprises a de novo amino acid sequence thatshares less than 10% sequence homology to the target antigen. In someembodiments, P₁ or P₂ is identified from a peptide library that containsrandom amino acid sequences.

In some embodiments, P₁ or P₂ comprises a peptide sequence of at least 5amino acids in length. In some embodiments, P₁ or P₂ comprises a peptidesequence of at least 6 amino acids in length. In some embodiments, P₁ orP₂ comprises a peptide sequence of at least 10 amino acids in length. Insome embodiments, P₁ or P₂ comprises a peptide sequence of at least 10amino acids in length and no more than 20 amino acids in length. In someembodiments, P₁ or P₂ comprises a peptide sequence of at least 16 aminoacids in length. In some embodiments, P₁ or P₂ comprises a peptidesequence of no more than 40 amino acids in length. In some embodiments,P₁ or P₂ comprises at least two cysteine amino acid residues. In someembodiments, P₁ or P₂ comprises a cyclic peptide or a linear peptide. Insome embodiments, P₁ or P₂ comprises a cyclic peptide. In someembodiments, P₁ or P₂ comprises a linear peptide.

In some embodiments, P₁ or P₂ comprise a modified amino acid ornon-natural amino acid, or a modified non-natural amino acid, or acombination thereof. In some embodiments, the modified amino acid or amodified non-natural amino acid comprises a post-translationalmodification. In some embodiments P₁ or P₂ comprise a modificationincluding, but not limited to acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent crosslinks, formation ofcystine, formation of pyroglutamate, formylation, gamma carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. Modifications are made anywhere to P₁or P₂ including the peptide backbone, the amino acid side chains, andthe terminus.

In some embodiments, P₁ or P₂ does not comprise albumin or an albuminfragment. In some embodiments, P₁ or P₂ does not comprise an albuminbinding domain.

A₁ and A₂

In some embodiments, A₁ or A₂ is an antigen recognizing molecule. Insome embodiments, the antigen recognizing molecule is an antibody or anantibody fragment. In some embodiments, the antibody or the antibodyfragment thereof comprises a single chain variable fragment, a singledomain antibody, Fab, Fab′. In some embodiments, the antibody orantibody fragment thereof comprises a single chain variable fragment(scFv), a heavy chain variable domain (VH domain), a light chainvariable domain (VL domain), or a variable domain (VHH) of a camelidderived single domain antibody. In some embodiments, the antibody orantibody fragment thereof comprises a single-chain variable fragment. Insome embodiments, the antibody or antibody fragment thereof is humanizedor human.

In some embodiments, A₁ or A₂ is a Fab. In some embodiments, the Fabcomprises (a) a Fab light chain polypeptide; and (b) a Fab heavy chainpolypeptide. In some embodiments, L₁ or L₂ is bound to N-terminus of theFab light chain polypeptide. In some embodiments L₁ or L₂ is bound toN-terminus of the Fab heavy chain polypeptide. In some embodiments, L₁or L₂ is bound to C-terminus of the Fab light chain polypeptide. In someembodiments, L₁ or L₂ is bound to C-terminus of the Fab heavy chainpolypeptide.

In some embodiments, A₁ or A₂ is a single chain variable fragment(scFv). In some embodiments, L₁ or L₂ is bound to N-terminus of thescFv. In some embodiments, L₁ or L₂ is bound to C-terminus of the scFv.In some embodiments, the scFv comprises a light chain variable domainand a heavy chain variable domain. In some embodiments, L₁ or L₂ isbound to a N-terminus of the light chain variable domain of the singlechain variable fragment (scFv). In some embodiments, L₁ or L₂ is boundto a N-terminus of the heavy chain variable domain of the single chainvariable fragment (scFv).

In some embodiments, the antibody or antibody fragment thereof comprisesan epidermal growth factor receptor (EGFR) binding domain. In someembodiments, the antibody or antibody fragment thereof comprises acluster of differentiation 3 (CD3) binding domain. In some embodiments,the antibody or antibody fragment thereof comprises a cluster ofdifferentiation 3 epsilon (CD3e) binding domain. In some embodiments,the target antigen comprises EGFR. In some embodiments, the targetantigen comprises CD3. In some embodiments, the target antigen comprisesCD3ε.

In some embodiments, A₁ or A₂ binds to a polypeptide that is part of aTCR-CD3 complex on the effector cell. In some embodiments, the targetantigen is an anti-CD3 effector cell antigen. In some embodiments, thepolypeptide that is part of the TCR-CD3 complex is human CD3ε. In someembodiments, A₁ or A₂ comprises an anti-CD3e single-chain variablefragment. In some embodiments, A₁ or A₂ comprises an anti-CD3esingle-chain variable fragment that has a K_(D) binding of 1 μM or lessto CD3 on CD3 expressing cells. In some embodiments, A_(t) or A₂comprises a variable light chain and variable heavy chain each of whichis capable of specifically binding to human CD3. In some embodiments, A₁or A₂ comprises complementary determining regions (CDRs) selected fromthe group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4),teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030(BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32,SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6,OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16,and 15865v19.

In some embodiments, A₁ or A₂ is a soluble T cell receptor (TCR). NativeTCRs are transmembrane receptors expressed on the surface of T cellsthat recognize antigens bound to major histocompatibility complexmolecules (MHC). Native TCRs are heterodimeric and comprise an alphapolypeptide chain and a beta polypeptide chain linked through adisulfide bond. The alpha polypeptide chain and the beta polypeptidechain are expressed as part of a complex with accessory proteins whichinclude, for example, two CD3 epsilon polypeptides, one CD3 gammapolypeptide, one CD3 delta polypeptide, and two CD3 zeta polypeptides.When a TCR engages with a target antigen and MHC, the T cell isactivated resulting in a series of signaling events mediated byassociated enzymes, co-receptors, adapter molecules, and activated orreleased transcription factors.

In native TCRs, the alpha polypeptide chain and the beta polypeptidechain comprise an extracellular domain, a transmembrane domain, and acytoplasmic domain. Each extracellular domain comprises a variableregion (V), a joining region (J), and a constant region (C). Theconstant region is N-terminal to the transmembrane domain, and thetransmembrane domain is N-terminal to the cytoplasmic domain. Thevariable regions of both the alpha polypeptide chain and the betapolypeptide chain comprise three hypervariable or complementaritydetermining regions (CDRs). The beta polypeptide chain usually containsa short diversity region between the variable and joining regions. Thethree CDRs are embedded into a framework sequence, with one CDR beingthe hypervariable region named CDR3. The alpha chain variable region(Vα) and the beta chain variable region (Vβ) are of several types thatare distinguished by their framework sequences, CDR1 and CDR2 sequences,and a partly defined CDR3 sequence.

TCRs are described using the International Immunogenetics (IMGT) TCRnomenclature. The Vα in IMGT nomenclature is referred to by a unique“TRAV” number. In the same way, Vβ is referred to by a unique “TRBV”number. The corresponding joining and constant regions are referred toas TRAJ and TRAC, respectively for the α joining and constant regions,and TRBJ and TRBC, respectively for the β joining and constant regions.The sequences defined by the IMGT nomenclature are known in the art andare contained within the online IMGT public database.

In some embodiments, the soluble TCR is a single chain TCR comprising avariable region of a TCR alpha extracellular domain, or fragmentthereof, and a variable region of a TCR beta extracellular domain, orfragment thereof. In some embodiments, the soluble TCR comprises analpha TCR polypeptide comprising a TCR alpha extracellular domain and abeta TCR polypeptide comprising a TCR beta extracellular domain.

In some embodiments, the soluble TCR is a single chain TCR comprising avariable region of a TCR alpha extracellular domain, or fragmentthereof, and a variable region of a TCR beta extracellular domain, orfragment thereof. In some embodiments, the soluble TCR comprises analpha TCR polypeptide comprising a TCR alpha extracellular domain and abeta TCR polypeptide comprising a TCR beta extracellular domain. In someembodiments, L₁ is bound to N-terminus of the alpha TCR polypeptide. Insome embodiments, L₁ is bound to N-terminus of the beta TCR polypeptide.In some embodiments, A₂ is bound to C-terminus of the alpha TCRpolypeptide. In some embodiments, A₂ is bound to N-terminus of the alphaTCR polypeptide. In some embodiments, A₂ is bound to C-terminus of thebeta TCR polypeptide. In some embodiments, A₂ is bound to N-terminus ofthe beta TCR polypeptide. In some embodiments, L₁ is bound to N-terminusof the alpha TCR polypeptide and A₂ is bound to N-terminus of the betaTCR polypeptide. In some embodiments, L₁ is bound to N-terminus of thealpha TCR polypeptide and A₂ is bound to C-terminus of the beta TCRpolypeptide. In some embodiments, L₁ is bound to N-terminus of the alphaTCR polypeptide and A₂ is bound to C-terminus of the alpha TCRpolypeptide. In some embodiments, L₁ is bound to N-terminus of the betaTCR polypeptide and A₂ is bound to N-terminus of the alpha TCRpolypeptide. In some embodiments, L₁ is bound to N-terminus of the betaTCR polypeptide and A₂ is bound to C-terminus of the beta TCRpolypeptide. In some embodiments, L₁ is bound to N-terminus of the betaTCR polypeptide and A₂ is bound to C-terminus of the alpha TCRpolypeptide.

In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen ascompared to the binding affinity for the target antigen or second targetantigen of a polypeptide or polypeptide complex that does not have P₁ orP₂ or L₁ or L₂. In some embodiments, the polypeptide or polypeptidecomplex has weaker binding affinity for the target antigen or secondtarget antigen that is at least 5× higher than the binding affinity forthe target antigen of a form of the polypeptide or polypeptide complexthat does not have P₁ or P₂ or L₁ or L₂. In some embodiments, thepolypeptide or polypeptide complex has weaker binding affinity for thetarget antigen or second target antigen that is at least 8× higher thanthe binding affinity for the target antigen or second target antigen ofa form of the polypeptide or polypeptide complex that does not have P₁or P₂ or L₁ or L₂. In some embodiments, the polypeptide or polypeptidecomplex has weaker binding affinity for the target antigen or secondtarget antigen that is at least 10× higher than the binding affinity forthe target antigen or second target antigen of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for the target antigen or second target antigen that is atleast 20× higher than the binding affinity for the target antigen orsecond target antigen of a form of the polypeptide or polypeptidecomplex that does not have P₁ or P₂ or L₁ or L₂. In some embodiments,the polypeptide or polypeptide complex has weaker binding affinity forthe target antigen or second target antigen that is at least 25× higherthan the binding affinity for the target antigen or second targetantigen of a form of the polypeptide or polypeptide complex that doesnot have P₁ or P₂ or L₁ or L₂. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 30× higher than the bindingaffinity for the target antigen or second target antigen of a form ofthe polypeptide or polypeptide complex that does not have P₁ or P₂ or L₁or L₂. In some embodiments, the polypeptide or polypeptide complex hasweaker binding affinity for the target antigen or second target antigenthat is at least 40× higher than the binding affinity for the targetantigen or second target antigen of a form of the polypeptide orpolypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for the target antigen or second target antigen that is atleast 50× higher than the binding affinity for the target antigen orsecond target antigen of a form of the polypeptide or polypeptidecomplex that does not have P₁ or P₂ or L₁ or L₂. In some embodiments,the polypeptide or polypeptide complex has weaker binding affinity forthe target antigen or second target antigen that is at least 60× higherthan the binding affinity for the target antigen or second targetantigen of a form of the polypeptide or polypeptide complex that doesnot have P₁ or P₂ or L₁ or L₂. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 70× higher than the bindingaffinity for the target antigen or second target antigen of a form ofthe polypeptide or polypeptide complex that does not have P₁ or P₂ or L₁or L₂. In some embodiments, the polypeptide or polypeptide complex hasweaker binding affinity for the target antigen or second target antigenthat is at least 75× higher than the binding affinity for the targetantigen or second target antigen of a form of the polypeptide orpolypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for the target antigen or second target antigen that is atleast 80× higher than the binding affinity for the target antigen orsecond target antigen of a form of the polypeptide or polypeptidecomplex that does not have P₁ or P₂ or L₁ or L₂. In some embodiments,the polypeptide or polypeptide complex has weaker binding affinity forthe target antigen or second target antigen that is at least 90× higherthan the binding affinity for the target antigen or second targetantigen of a form of the polypeptide or polypeptide complex that doesnot have P₁ or P₂ or L₁ or L₂. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 100× higher than the bindingaffinity for the target antigen or second target antigen of a form ofthe polypeptide or polypeptide complex that does not have P₁ or P₂ or L₁or L₂. In some embodiments, the polypeptide or polypeptide complex hasweaker binding affinity for the target antigen or second target antigenthat is at least 120× higher than the binding affinity for the targetantigen or second target antigen of a form of the polypeptide orpolypeptide complex that does not have P₁ or P₂ or L₁ or L₂.

In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen ascompared to the binding affinity for the target antigen or second targetantigen of the polypeptide or polypeptide complex in which L₁ or L₂ hasbeen cleaved. In some embodiments, the polypeptide or polypeptidecomplex has weaker binding affinity for the target antigen or secondtarget antigen that is at least 5× higher than the binding affinity forthe target antigen or second target antigen of the polypeptide orpolypeptide complex in which L₁ or L₂ has been cleaved. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for the target antigen or second target antigen that is atleast 8× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 10× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen that isat least 20× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 25× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen that isat least 30× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 40× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen that isat least 50× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 60× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen that isat least 70× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 75× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen that isat least 80× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 90× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for the target antigen or second target antigen that isat least 100× higher than the binding affinity for the target antigen orsecond target antigen of the polypeptide or polypeptide complex in whichL₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for the target antigenor second target antigen that is at least 120× higher than the bindingaffinity for the target antigen or second target antigen of thepolypeptide or polypeptide complex in which L₁ or L₂ has been cleaved.In some embodiments, L₁ or L₂ is cleaved by a protease. In someembodiments, the protease comprises a tumor specific protease. In someembodiments, the protease comprises a matrix metalloprotease (MMP) or aserine protease. In some embodiments, the matrix metalloproteasecomprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, theserine protease comprises matriptase, urokinase, or hepsin.

In some embodiments, A₁ or A₂ is a cytokine or cytokine fragment. Insome embodiments, A₁ or A₂ is a mutein of the cytokine or the cytokinefragment. In some embodiments, the cytokine or the cytokine fragment isa mutein of the cytokine or the cytokine fragment.

Cytokines are a diverse group of small peptides, including chemokines,interferons, interleukins, lymphokines, adipokines, mesenchymal growthfactors, and tumor necrosis factors, which are involved in intercellularsignaling in a variety of biological pathways. They are particularlyimportant in immune and inflammatory responses. Signaling occursfollowing recognition of the cytokine by a corresponding cytokinereceptor, which are transmembrane receptors comprising an extracellulardomain for ligand binding and an intracellular domain that allows signaltransduction.

The diversity of cytokines comes with a corresponding diversity incytokine receptors, which can comprise a single chain or subunit ordimeric/multimeric domains. Cytokine receptors include Type I cytokinereceptors, exemplified by interleukin receptors, and Type II cytokinereceptors, exemplified by interferon receptors, both of which comprise acytokine receptor homology domain (CHD). The CHD of Type I cytokinereceptors share a common amino acid motif (WSXWS (SEQ ID NO: 27)), whileType II cytokine receptors lack this motif. Cytokine receptors caninclude an alpha subunit, beta subunit, gamma subunit, or dimeric, ortrimeric combinations thereof. In one example, a high affinity receptorfor IL-2 comprises an IL-2Rα subunit, IL-2Rβ subunit, and IL-2Rγsubunit, an intermediate affinity receptor for IL-2 comprises only theIL-2Rβ subunit and IL-2Rγ subunit, and low affinity receptor for IL-2comprises only the IL-2Rα subunit.

In some embodiments, the cytokine is a chemokine, an interferon, aninterleukin, a lymphokine, an adipokine, a growth factor, or a tumornecrosis factor. In some embodiments, the interferon (IFN) is IFNα,IFNβ, IFNγ, or a fragment thereof. In some embodiments, the interleukin(IL) is IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or afragment thereof. In some embodiments, the growth factor isgranulocyte-macrophage colony-stimulating factor (GM-CSF) or a fragmentthereof. In some embodiments the cytokine is TGF-γ.

In some embodiments, a cytokine mutein is a variant of a wild-typecytokine. In some embodiments, a cytokine mutein is a mutant of awild-type cytokine. In some embodiments, the cytokine mutein comprisesat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50amino acid substitutions relative to a wild-type cytokine. In someembodiments, the cytokine mutein comprises no more than 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutionsrelative to a wild-type cytokine. In some embodiments, the cytokinemutein is a non-naturally occurring cytokine. In some embodiments, thecytokine mutein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or more than 50 amino acid substitutions relative to anaturally occurring cytokine. In some embodiments, the cytokine muteincomprises no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, ormore than 50 amino acid substitutions relative to a naturally occurringcytokine.

In some embodiments, the cytokine or cytokine fragment binds to acytokine receptor. In some embodiments, the cytokine receptor is areceptor for a chemokine, an interferon, an interleukin, a lymphokine,an adipokine, a growth factor, or a tumor necrosis factor. In someembodiments, the cytokine receptor is a type I cytokine receptor or atype II cytokine receptor. In some embodiments, the cytokine receptor isa dimer or a trimer. In some embodiments, the cytokine receptorcomprises an alpha subunit, a beta subunit, a gamma subunit, or anycombination thereof. For example, in some embodiments, the cytokinereceptor comprises an alpha subunit, a beta subunit, and a gammasubunit. In another example, in some embodiments, the cytokine receptorcomprises a beta subunit and a gamma subunit. In some embodiments, thecytokine receptor comprises an alpha subunit and a beta subunit.

In some embodiments, the polypeptide or polypeptide complex has a weakerbinding affinity for its cytokine receptor as compared to the bindingaffinity for the cytokine receptor of a polypeptide or polypeptidecomplex that does not have P₁ or P₂ or L₁ or L₂. In some embodiments,the polypeptide or polypeptide complex has weaker binding affinity forits cytokine receptor that is at least 5× weaker than the bindingaffinity for the cytokine receptor of a form of the polypeptide orpolypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 8× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 10× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 20× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 25× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 30× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 40× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 50× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 60× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 70× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 75× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 80× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 90× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 100× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 120× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the polypeptide or polypeptide complex has weaker bindingaffinity for its cytokine receptor that is at least 150× weaker than thebinding affinity for the cytokine receptor of a form of the polypeptideor polypeptide complex that does not have P₁ or P₂ or L₁ or L₂. In someembodiments, the cytokine or cytokine fragment comprises an interferon,GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, thecytokine or cytokine fragment comprises IL-2, IL-12, IL-6, IL-4, IL-10,or TGF-3. In some embodiments, the cytokine receptor comprises aninterferon receptor, GM-CSF receptor, IL-2 receptor, IL-7 receptor,IL-12 receptor, IL-15 receptor, or IL-21 receptor. In some embodiments,the cytokine receptor comprises IL-2 receptor, IL-12 receptor, IL-6receptor, IL-4 receptor, IL-10 receptor, or TGF-β receptor.

In some embodiments, the polypeptide or polypeptide complex has weakerbinding affinity for its cytokine receptor as compared to the bindingaffinity for the cytokine receptor of the polypeptide or polypeptidecomplex in which L₁ or L₂ has been cleaved. In some embodiments, thepolypeptide or polypeptide complex has weaker binding affinity for itscytokine receptor that is at least 5× weaker than the binding affinityfor the cytokine receptor of the polypeptide or polypeptide complex inwhich L₁ or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 8× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 10× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 15× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 20× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 25× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 30× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 40× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 50× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 60× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 70× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 75× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 80× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 90× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the polypeptide orpolypeptide complex has weaker binding affinity for its cytokinereceptor that is at least 100× weaker than the binding affinity for thecytokine receptor of the polypeptide or polypeptide complex in which L₁or L₂ has been cleaved. In some embodiments, the cytokine or cytokinefragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, orIL-21. In some embodiments, the cytokine receptor comprises aninterferon receptor, GM-CSF receptor, IL-2 receptor, IL-7 receptor,IL-12 receptor, IL-15 receptor, or IL-21 receptor. In some embodiments,L₁ or L₂ is cleaved by a protease. In some embodiments, the proteasecomprises a tumor specific protease. In some embodiments, the proteasecomprises a matrix metalloprotease (MMP) or a serine protease. In someembodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9,MMP13, or MMP14. In some embodiments, the serine protease comprisesmatriptase, urokinase, or hepsin.

Half-Life Extending Moiety

In some embodiments, P₁ is further linked to a half-life extendingmoiety. In some embodiments, P₁ is further linked to a half-lifeextending moiety in a configuration according to Formula Ia

A₁-L₁-P₁-L₃-H₁  (Formula Ia)

wherein H₁ is the half-life extending moiety and L₃ is a linker thatconnects H₁ to P₁. In some embodiments, L₃ is a non-cleavable linker. Insome embodiments, the half-life extending moiety (H₁) does not block A₁binding to the target antigen. In some embodiments, the half-lifeextending moiety (H₁) does not have binding affinity to A₁. In someembodiments, the half-life extending moiety (H₁) does not have bindingaffinity to the target antigen. In some embodiments, the half-lifeextending moiety (H₁) does not shield A₁ from the target antigen. Insome embodiments, the half-life extending moiety (H₁) is not directlylinked to A₁.

In some embodiments, the half-life extending moiety (H₁) does not blockA₁ binding to the cytokine receptor. In some embodiments, the half-lifeextending moiety (H₁) does not have binding affinity to the cytokine orcytokine receptor. In some embodiments, the half-life extending moiety(H₁) does not shield the cytokine or cytokine fragment from the cytokinereceptor. In some embodiments, the half-life extending moiety (H₁) isnot directly linked to the cytokine or cytokine fragment.

In some embodiments, H₁ comprises an amino acid sequence that hasrepetitive sequence motifs. In some embodiments, H₁ comprises an aminoacid sequence that has highly ordered secondary structure. “Highlyordered secondary structure,” as used in this context, means that atleast about 50%, or about 70%, or about 80%, or about 90%, of amino acidresidues of H₁ contribute to secondary structure, as measured ordetermined by means, including, but not limited to, spectrophotometry(e.g. by circular dichroism spectroscopy in the “far-UV” spectral region(190-250 nm), and computer programs or algorithms, such as theChou-Fasman algorithm and the Gamier-Osguthorpe-Robson (“GOR”)algorithm.

In some embodiments, H₁ comprises a polymer. In some embodiments, thepolymer is polyethylene glycol (PEG). In some embodiments, H₁ comprisesalbumin. In some embodiments, H₁ comprises a Fc domain. In someembodiments, the albumin is serum albumin. In some embodiments, thealbumin is human serum albumin. In some embodiments, H₁ comprises apolypeptide, a ligand, or a small molecule. In some embodiments, thepolypeptide, the ligand or the small molecule binds serum protein or afragment thereof, a circulating immunoglobulin or a fragment thereof, orCD35/CR1. In some embodiments, the serum protein comprises athyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, atransferrin, transferrin receptor or a transferrin-binding portionthereof, a fibrinogen, or an albumin. In some embodiments, thecirculating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4,s1gA, IgM or IgD. In some embodiments, the serum protein is albumin. Insome embodiments, the polypeptide is an antibody. In some embodiments,the antibody comprises a single domain antibody, a single chain variablefragment or a Fab. In some embodiments, the antibody comprises a singledomain antibody. In some embodiments, the antibody comprises a singledomain antibody that binds to albumin. In some embodiments, the antibodycomprises a single domain antibody that binds to human serum albumin. Insome embodiments, the antibody is a human or humanized antibody. In someembodiments, the single domain antibody is selected from the groupconsisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or afragment thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G,10GE, and SA21.

In some embodiments, H₁ comprises a single domain antibody. In someembodiments, H₁ comprises a single domain antibody that binds toalbumin. In some embodiments, H₁ comprises a single domain antibody thatbinds to human serum albumin.

In some embodiments, H₁ comprise a modified amino acid or non-naturalamino acid, or a modified non-natural amino acid, or a combinationthereof. In some embodiments, the modified amino acid or a modifiednon-natural amino acid comprises a post-translational modification. Insome embodiments, H₁ comprise a modification including, but not limitedto acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent crosslinks, formation ofcystine, formation of pyroglutamate, formylation, gamma carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. Modifications are made anywhere to H₁including the peptide backbone, the amino acid side chains, and theterminus.

Polynucleotides Encoding Polypeptides or Polypeptide Complexes

Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding polypeptides or polypeptide complexes asdisclosed herein. Described herein, in some embodiments, are isolatedrecombinant nucleic acid molecules encoding polypeptides comprising acleavable linker.

As disclosed herein, in some embodiments, are isolated recombinantnucleic acid molecules encoding polypeptides comprising a cleavablelinker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG).

In some embodiments, the cleavable linker comprises the amino acidsequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, thecleavable linker comprises the amino acid sequence of SEQ ID NO: 26(AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprisesthe amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In someembodiments, the cleavable linker comprises the amino acid sequence ofSEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the cleavable linkercomprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding an isolated polypeptide comprising a cleavablelinker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG)(SEQ ID NO: 3), Linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4), Linker 3(SPLGLSGRSDAG) (SEQ ID NO: 5), or Linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO:6), or an isolated polypeptide comprising a cleavable linker that has 1,2, or 3 amino acid substitutions, additions, or deletions relative tothe amino acid sequence of Linker 1, Linker 2, Linker 3, or Linker 4.Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding an isolated polypeptide comprising a cleavablelinker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG)(SEQ ID NO: 3). Disclosed herein, in some embodiments, are isolatedrecombinant nucleic acid molecules encoding an isolated polypeptidecomprising a cleavable linker according to the amino acid sequence ofLinker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4). Disclosed herein, in someembodiments, are isolated recombinant nucleic acid molecules encoding anisolated polypeptide comprising a cleavable linker according to theamino acid sequence of Linker 3 (SPLGLSGRSDAG) (SEQ ID NO: 5). Disclosedherein, in some embodiments, are isolated recombinant nucleic acidmolecules encoding an isolated polypeptide comprising a cleavable linkeraccording to the amino acid sequence of Linker 4 (LSGRSDAGSPLGLAG) (SEQID NO: 6).

Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding an isolated polypeptide comprising a cleavablelinker according to the amino acid sequence LSGRSDAG (SEQ ID NO: 1).

Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding polypeptides or polypeptide complexes accordingto Formula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ comprises the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁comprises the cleavable linker; and P₁ comprises a peptide that impairsbinding of the antigen binding domain to the target antigen or impairsbinding of the cytokine to the cytokine receptor.

Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding polypeptides or polypeptide complexes comprisingFormula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ comprises the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁comprises the cleavable linker; and P₁ comprises a peptide that impairsbinding of the antigen binding domain to the target antigen or impairsbinding of the cytokine to the cytokine receptor. Disclosed herein, insome embodiments, are isolated recombinant nucleic acid moleculesencoding polypeptides or polypeptide complexes according to Formula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ is the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁ is thecleavable linker; and P₁ is a peptide that impairs binding of theantigen binding domain to the target antigen or impairs binding of thecytokine to the cytokine receptor. Disclosed herein, in someembodiments, are isolated recombinant nucleic acid molecules encodingpolypeptides or polypeptide complexes comprising Formula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ is the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁ is thecleavable linker; and P₁ is a peptide that impairs binding of theantigen binding domain to the target antigen or impairs binding of thecytokine to the cytokine receptor.

Disclosed herein, in some embodiments, are polypeptides or polypeptidecomplexes, wherein the isolated polypeptide is complexed with a secondisolated polypeptide comprising a second antigen binding domain or asecond cytokine. Disclosed herein, in some embodiments, are isolatedrecombinant nucleic acid molecules encoding polypeptides or polypeptidecomplexes according to Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ comprises the second antigen binding domain or the secondcytokine; L₂ comprises a second cleavable linker; and P₂ comprises asecond peptide that impairs binding of the second antigen binding domainto a second target antigen or impairs binding of the second cytokine toa second cytokine receptor. Disclosed herein, in some embodiments, areisolated recombinant nucleic acid molecules encoding polypeptides orpolypeptide complexes comprising Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ comprises the second antigen binding domain or the secondcytokine; L₂ comprises a second cleavable linker; and P₂ comprises asecond peptide that impairs binding of the second antigen binding domainto a second target antigen or impairs binding of the second cytokine toa second cytokine receptor. Disclosed herein, in some embodiments, areisolated recombinant nucleic acid molecules encoding polypeptides orpolypeptide complexes according to Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ is the second antigen binding domain or the second cytokine;L₂ is a second cleavable linker; and P₂ is a second peptide that impairsbinding of the second antigen binding domain to a second target antigenor impairs binding of the second cytokine to a second cytokine receptor.Disclosed herein, in some embodiments, are isolated recombinant nucleicacid molecules encoding polypeptides or polypeptide complexes comprisingFormula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ is the second antigen binding domain or the second cytokine;L₂ is a second cleavable linker; and P₂ is a second peptide that impairsbinding of the second antigen binding domain to a second target antigenor impairs binding of the second cytokine to a second cytokine receptor.

Pharmaceutical Compositions

Disclosed herein, in some embodiments, are pharmaceutical compositionscomprising: (a) the polypeptides or polypeptide complexes as disclosedherein; and (b) a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG) and (b)a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG) and(b) a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of SEQ ID NO: 26(AGLLAPPGGLSGRSDAG) and (b) a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of SEQ ID NO: 4(AAGLLAPPGGLSGRSDAG) and (b) a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG) and(b) a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG)and (b) a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) (SEQ IDNO: 3), Linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4), Linker 3(SPLGLSGRSDAG) (SEQ ID NO: 5), or Linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO:6), or the polypeptides or polypeptide complexes comprising a cleavablelinker that has 1, 2, or 3 amino acid substitutions, additions, ordeletions relative to the amino acid sequence of Linker 1, Linker 2,Linker 3, or Linker 4 and (b) a pharmacetically acceptable excipient. Insome embodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) (SEQ IDNO: 3) and (b) a pharmacetically acceptable excipient. In someembodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of Linker 2 (AAGLLAPPGGLSGRSDAG)(SEQ ID NO: 4) and (b) a pharmacetically acceptable excipient. In someembodiments, the pharmaceutical composition comprises (a thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of Linker 3 (SPLGLSGRSDAG) (SEQ IDNO: 5) and (b) a pharmacetically acceptable excipient. In someembodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the amino acid sequence of Linker 4 (LSGRSDAGSPLGLAG) (SEQID NO: 6) and (b) a pharmacetically acceptable excipient. In someembodiments, the pharmaceutical composition comprises (a) thepolypeptides or polypeptide complexes comprising a cleavable linkeraccording to the the amino acid sequence LSGRSDAG (SEQ ID NO: 1) and (b)a pharmacetically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a)isolated polypeptide polypeptides or polypeptide complexes according toFormula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ comprises the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁comprises the cleavable linker; and P₁ comprises a peptide that impairsbinding of the antigen binding domain to the target antigen or impairsbinding of the cytokine to the cytokine receptor; and (b) apharmacetically acceptable excipient. In some embodiments, thepharmaceutical composition comprises (a) isolated polypeptidepolypeptides or polypeptide complexes comprising Formula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ comprises the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁comprises the cleavable linker; and P₁ comprises a peptide that impairsbinding of the antigen binding domain to the target antigen or impairsbinding of the cytokine to the cytokine receptor; and (b) apharmacetically acceptable excipient. In some embodiments, thepharmaceutical composition comprises (a) isolated polypeptidepolypeptides or polypeptide complexes according to Formula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ is the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁ is thecleavable linker; and P₁ is a peptide that impairs binding of theantigen binding domain to the target antigen or impairs binding of thecytokine to the cytokine receptor; and (b) a pharmacetically acceptableexcipient. In some embodiments, the pharmaceutical composition comprises(a) isolated polypeptide polypeptides or polypeptide complexescomprising Formula I:

A₁-L₁-P₁  (Formula I)

wherein A₁ is the antigen binding domain that binds to the targetantigen or the cytokine that binds to the cytokine receptor; L₁ is thecleavable linker; and P₁ is a peptide that impairs binding of theantigen binding domain to the target antigen or impairs binding of thecytokine to the cytokine receptor; and (b) a pharmacetically acceptableexcipient.

Disclosed herein, in some embodiments, are polypeptides or polypeptidecomplexes, wherein the isolated polypeptide is complexed with a secondisolated polypeptide comprising a second antigen binding domain or asecond cytokine. In some embodiments, the pharmaceutical compositioncomprises (a) isolated polypeptide polypeptides or polypeptide complexesaccording to Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ comprises the second antigen binding domain or the secondcytokine; L₂ comprises a second cleavable linker; and P₂ comprises asecond peptide that impairs binding of the second antigen binding domainto a second target antigen or impairs binding of the second cytokine toa second cytokine receptor; and (b) a pharmacetically acceptableexcipient. In some embodiments, the pharmaceutical composition comprises(a) isolated polypeptide polypeptides or polypeptide complexescomprising Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ comprises the second antigen binding domain or the secondcytokine; L₂ comprises a second cleavable linker; and P₂ comprises asecond peptide that impairs binding of the second antigen binding domainto a second target antigen or impairs binding of the second cytokine toa second cytokine receptor; and (b) a pharmacetically acceptableexcipient. In some embodiments, the pharmaceutical composition comprises(a) isolated polypeptide polypeptides or polypeptide complexes accordingto Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ is the second antigen binding domain or the second cytokine;L₂ is a second cleavable linker; and P₂ is a second peptide that impairsbinding of the second antigen binding domain to a second target antigenor impairs binding of the second cytokine to a second cytokine receptor;and (b) a pharmacetically acceptable excipient. In some embodiments, thepharmaceutical composition comprises (a) isolated polypeptidepolypeptides or polypeptide complexes comprising Formula II:

A₂-L₂-P₂  (Formula II)

wherein A₂ is the second antigen binding domain or the second cytokine;L₂ is a second cleavable linker; and P₂ is a second peptide that impairsbinding of the second antigen binding domain to a second target antigenor impairs binding of the second cytokine to a second cytokine receptor;and (b) a pharmacetically acceptable excipient.

In some embodiments, the polypeptide or polypeptide complex furthercomprises a detectable label, a therapeutic agent, or a pharmacokineticmodifying moiety. In some embodiments, the detectable label comprises afluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or acontrast agent.

For administration to a subject, the polypeptide or polypeptide complexas disclosed herein, may be provided in a pharmaceutical compositiontogether with one or more pharmaceutically acceptable carriers orexcipients. The term “pharmaceutically acceptable carrier” includes, butis not limited to, any carrier that does not interfere with theeffectiveness of the biological activity of the ingredients and that isnot toxic to the patient to whom it is administered. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Preferably, thecompositions are sterile. These compositions may also contain adjuvantssuch as preservative, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents.

The pharmaceutical composition may be in any suitable form, (dependingupon the desired method of administration). It may be provided in unitdosage form, may be provided in a sealed container and may be providedas part of a kit. Such a kit may include instructions for use. It mayinclude a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by anyappropriate route, including a parenteral (e.g., subcutaneous,intramuscular, or intravenous) route. Such compositions may be preparedby any method known in the art of pharmacy, for example by mixing theactive ingredient with the carrier(s) or excipient(s) under sterileconditions.

Dosages of the substances of the present disclosure can vary betweenwide limits, depending upon the disease or disorder to be treated, theage and condition of the individual to be treated, etc. and a physicianwill ultimately determine appropriate dosages to be used.

Table 1 provides the amino acid sequences of constructs describedherein.

TABLE 1 Summary of Amino Acid Sequences Construct ConstructAmino Acid Sequence SEQ ID ID Description (N to C) NO: LINKER SEQUENCESLinker-0 Cleavable linker ISSGLLSGRSDNH  2 (control) Linker-1Cleavable linker ISSGLLSGRSDAG  3 Linker-2 Cleavable linkerAAGLLAPPGGLSGRSDAG  4 Linker-3 Cleavable linker SPLGLSGRSDAG  5 Linker-4Cleavable linker LSGRSDAGSPLGLAG  6 Linker-5 Cleavable linker LSGRSDAG 1 Linker-6 Cleavable linker AGLLAPPGGLSGRSDAG 26 PEPTIDE MASK SEQUENCESPeptide-1 anti-EGFR peptide QGQSGQLSCEGWAMNREQCRA  7 mask Peptide-2anti-EGFR peptide GGPCRSHIDVAKPICV  8 mask Peptide-3 anti-CD3 peptideQGQSGQGYLWGCEWNCGGITT  9 mask Peptide-4 anti-CD3 peptideQGQSGSGYLWGCEWNCAGITT 10 mask HALF-LIFE EXTENDING MOIETIES HE-110G single domain EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 11 antibodyQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS FULL LENGTH CONSTRUCTS PC-1Light Chain QGQSGQLSCEGWAMNREQCRAGSSGGSGGSGGSGISSG 12 Sequence: N-LLSGRSDNHGSSGTDILLTQSPVILSVSPGERVSFSCRASQ [Peptide-1]-[Linker-SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT 0]-[anti-EGFR FabDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR light chain]-CTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ (control)WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECPC-1 Heavy Chain EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 13Sequence: N-[10G QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTSDA]-[Peptide-3]- TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG[Linker-0]-[anti-CD3 GGGSGGGSQGQSGQGYLWGCEW scFv (light chain-NCGGITTGSSGGSGGSGGISSGLLSGRSDNHGGGSQTVVT heavy chain)]-[anti-QEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ EGFR Fab heavyAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPE chain]-CDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSG (control)GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGHHHHHHHHGGGLNDIFEAQKIEWHE PC-2 Light ChainGGPCRSHIDVAKPICVGGGGSGGSISSGLLSGRSDAGGGG 14 Sequence: N-SDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT [Peptide-2]-[Linker-NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDI 1]-[anti-EGFR FabADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSD light chain]-CEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC PC-2 Heavy ChainEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 15 Sequence: N-[10GQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT SDA]-[Peptide-4]-TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG [Linker-1]-[anti-CD3GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSGGS scFv (light chain-GGISSGLLSGRSDAGGGGSQTVVTQEPSLTVSPGGTVTLT heavy chain)]-[anti-CRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG EGFR Fab heavyTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNL chain]-CWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA HHHHHHHH PC-3 Light ChainGGPCRSHIDVAKPICVGGGGSSGGSAAGLLAPPGGLSGRS 16 Sequence: N-DAGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIH [Peptide-2]-[Linker-WYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSIN 2]-[anti-EGFR FabSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSV light chain]-CFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC PC-3Heavy Chain EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 17 Sequence: N-[10GQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT SDA]-[Peptide-4]-TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG [linker Linker-2]-GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSAA [anti-CD3 scFv (lightGLLAPPGGLSGRSDAGGGGSQTVVTQEPSLTVSPGGTVT chain-heavy chain)]-LTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA [anti-EGFR FabPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSN heavy chain]-CLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAA AHHHHHHHH PC-4 Light ChainGGPCRSHIDVAKPICVGGGGSGGGGSPLGLSGRSDAGGG 18 Sequence: N-GSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQR [Peptide-2]-[Linker-TNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESED 3]-[anti-EGFR FabIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS light chain]-CDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC PC-4Heavy Chain EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 19 Sequence: N-[10GQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT SDA]-[Peptide-4]-TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG [Linker-3]-[anti-CD3GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSGG scFv (light chain-GSGGSPLGLSGRSDAGGGGSQTVVTQEPSLTVSPGGTVT heavy chain)]-[anti-LTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA EGFR Fab heavyPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSN chain]-CLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAA AHHHHHHHH PC-5 Light ChainGGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAGS 20 Sequence: N-GGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQ [Peptide-2]-[Linker-RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESE 4]-[anti-EGFR FabDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPP light chain]-CSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC PC-5Heavy Chain EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 21 Sequence: N-[10GQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT SDA]-[Peptide-4]-TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG [Linker-4]-[anti-CD3GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSGGL scFv (light chain-SGRSDAGSPLGLAGSGGGSQTVVTQEPSLTVSPGGTVTL heavy chain)]-[anti-TCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP EGFR Fab heavyGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNL chain]-CWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA HHHHHHHH PC-6 N-[anti-EGFR FabQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN 22 heavy chain]-CGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC PC-6N-[anti-CD3 scFv QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ 23(light chain-heavy QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSchain)]-[anti-EGFR GVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGFab heavy chain]-C GGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGHHHHHHHHGGGLNDIFEAQKI EWHE PC-7 N-[anti-EGFR FabDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN 24 heavy chain]-CGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC PC-7N-[anti-CD3 scFv QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ 25(light chain-heavy QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSchain)]-[anti-EGFR GVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGFab heavy chain]-C GGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGHHHHHHHHGGGLNDIFEAQKI EWHE

Polypeptides or polypeptide complexes, in some embodiments, comprise asequence set forth in Table 1. In some embodiments, the sequencecomprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 4, 5,7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. Insome instances, the sequence comprises at least or about 95% homology toSEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 1⁹, 20, 21,22, 23, 24, or 25. In some instances, the sequence comprises at least orabout 97% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some instances, thesequence comprises at least or about 99% homology to SEQ ID NOs: 1, 2,4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25. In some instances, the sequence comprises at least or about 100%homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25. In some instances, the sequence comprisesat least a portion having at least or about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, or more than 400 amino acids of SEQ ID NOs: 1, 2, 4,5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

Percent (%) sequence identity with respect to a reference polypeptidesequence is the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in thereference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity.

Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are known for instance,using publicly available computer software such as BLAST, BLAST-2, ALIGNor Megalign (DNASTAR) software. Appropriate parameters for aligningsequences are able to be determined, including algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For purposes herein, however, % amino acid sequence identityvalues are generated using the sequence comparison computer programALIGN-2. The ALIGN-2 sequence comparison computer program was authoredby Genentech, Inc., and the source code has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

Production of Polypeptides Comprising Cleavable Linkers

In some embodiments, polypeptides described herein (e.g., antibodies andits binding fragments) are produced using any method known in the art tobe useful for the synthesis of polypeptides (e.g., antibodies), inparticular, by chemical synthesis or by recombinant expression, and arepreferably produced by recombinant expression techniques.

In some instances, an antibody or its binding fragment thereof isexpressed recombinantly, and the nucleic acid encoding the antibody orits binding fragment is assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody isoptionally generated from a suitable source (e.g., an antibody cDNAlibrary, or cDNA library generated from any tissue or cells expressingthe immunoglobulin) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

In some instances, an antibody or its binding is optionally generated byimmunizing an animal, such as a mouse, to generate polyclonal antibodiesor, more preferably, by generating monoclonal antibodies, e.g., asdescribed by Kohler and Milstein (1975, Nature 256:495-497) or, asdescribed by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al.(1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Alternatively, a clone encoding at least the Fab portion ofthe antibody is optionally obtained by screening Fab expressionlibraries (e.g., as described in Huse et al., 1989, Science246:1275-1281) for clones of Fab fragments that bind the specificantigen or by screening antibody libraries (See, e.g., Clackson et al.,1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA94:4937).

In some embodiments, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity are used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region.

In some embodiments, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted toproduce single chain antibodies. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide. Techniquesfor the assembly of functional Fv fragments in E. coli are alsooptionally used (Skerra et al., 1988, Science 242:1038-1041).

In some embodiments, an expression vector comprising the nucleotidesequence of an antibody or the nucleotide sequence of an antibody istransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation), and the transfected cells are then cultured byconventional techniques to produce the antibody. In specificembodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

In some embodiments, a variety of host-expression vector systems isutilized to express an antibody, or its binding fragment describedherein. Such host-expression systems represent vehicles by which thecoding sequences of the antibody is produced and subsequently purified,but also represent cells that are, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibody or itsbinding fragment in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing an antibody or its binding fragmentcoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing an antibody or itsbinding fragment coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing anantibody or its binding fragment coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anantibody or its binding fragment coding sequences; or mammalian cellsystems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. In some instances, cell lines that stablyexpress an antibody are optionally engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellsare transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells are thenallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turnare cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express the antibody or its bindingfragments.

In some instances, a number of selection systems are used, including butnot limited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genesare employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance are used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

In some instances, the expression levels of an antibody are increased byvector amplification (for a review, see Bebbington and Hentschel, theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing an antibodyis amplifiable, an increase in the level of inhibitor present in cultureof host cell will increase the number of copies of the marker gene.Since the amplified region is associated with the nucleotide sequence ofthe antibody, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification of anantibody is used, for example, by chromatography (e.g., ion exchange,affinity, particularly by affinity for the specific antigen afterProtein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Expression Vectors

In some embodiments, vectors include any suitable vectors derived fromeither a eukaryotic or prokaryotic sources. In some cases, vectors areobtained from bacteria (e.g. E. coli), insects, yeast (e.g. Pichiapastoris), algae, or mammalian sources. Exemplary bacterial vectorsinclude pACYC177, pASK75, pBAD vector series, pBADM vector series, pETvector series, pETM vector series, pGEX vector series, pHAT, pHAT2,pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAGShift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET,pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b,pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12,FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such aspPolh-MAT1, or pPolh-MAT2.

In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway®pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector,Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector,pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA,B, & C Pichiapastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector,pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeastvector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.

Exemplary algae vectors include pChlamy-4 vector or MCS vector.

Examples of mammalian vectors include transient expression vectors orstable expression vectors. Mammalian transient expression vectors mayinclude pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23,pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c,p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1,pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stableexpression vector may include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10,p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, orpBICEP-CMV 2.

In some instances, a cell-free system is a mixture of cytoplasmic and/ornuclear components from a cell and is used for in vitro nucleic acidsynthesis. In some cases, a cell-free system utilizes either prokaryoticcell components or eukaryotic cell components. Sometimes, a nucleic acidsynthesis is obtained in a cell-free system based on for exampleDrosophila cell, Xenopus egg, or HeLa cells. Exemplary cell-free systemsinclude, but are not limited to, E. coli S30 Extract system, E. coli T7S30 system, or PURExpress®.

Host Cells

In some embodiments, a host cell includes any suitable cell such as anaturally derived cell or a genetically modified cell. In someinstances, a host cell is a production host cell. In some instances, ahost cell is a eukaryotic cell. In other instances, a host cell is aprokaryotic cell. In some cases, a eukaryotic cell includes fungi (e.g.,yeast cells), animal cell or plant cell. In some cases, a prokaryoticcell is a bacterial cell. Examples of bacterial cell includegram-positive bacteria or gram-negative bacteria. Sometimes thegram-negative bacteria is anaerobic, rod-shaped, or both.

In some instances, gram-positive bacteria include Actinobacteria,Firmicutes or Tenericutes. In some cases, gram-negative bacteria includeAquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi/Bacteroidetes(FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae,Planctomycetes-Verrucomicrobia/Chlamydiae (PVC group), Proteobacteria,Spirochaetes or Synergistetes. Other bacteria can be Acidobacteria,Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres,Dictyoglomi, Thermodesulfobacteria or Thermotogae. A bacterial cell canbe Escherichia coli, Clostridium botulinum, or Coli bacilli.

Exemplary prokaryotic host cells include, but are not limited to, BL21,Machl™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110,TOP10F′, INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, orStbl4™.

In some instances, animal cells include a cell from a vertebrate or froman invertebrate. In some cases, an animal cell includes a cell from amarine invertebrate, fish, insects, amphibian, reptile, or mammal. Insome cases, a fungus cell includes a yeast cell, such as brewer's yeast,baker's yeast, or wine yeast.

Fungi include ascomycetes such as yeast, mold, filamentous fungi,basidiomycetes, or zygomycetes. In some instances, yeast includesAscomycota or Basidiomycota. In some cases, Ascomycota includesSaccharomycotina (true yeasts, e.g. Saccharomyces cerevisiae (baker'syeast)) or Taphrinomycotina (e.g. Schizosaccharomycetes (fissionyeasts)). In some cases, Basidiomycota includes Agaricomycotina (e.g.Tremellomycetes) or Pucciniomycotina (e.g. Microbotryomycetes).

Exemplary yeast or filamentous fungi include, for example, the genus:Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula,Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi,Aspergillus, Fusarium, or Trichoderma. Exemplary yeast or filamentousfungi include, for example, the species: Saccharomyces cerevisiae,Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candidaalbicans, Candida tropicalis, Candida stellatoidea, Candida glabrata,Candida krusei, Candida parapsilosis, Candida guilliermondii, Candidaviswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichiametanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenulapolymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowialipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillusniger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae,Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis,Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii,Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii,or Saccharomyces boulardii.

Exemplary yeast host cells include, but are not limited to, Pichiapastoris yeast strains such as GS115, KM71H₁, SMD1168, SMD1 168H, andX-33; and Saccharomyces cerevisiae yeast strain such as INVSc1.

In some instances, additional animal cells include cells obtained from amollusk, arthropod, annelid or sponge. In some cases, an additionalanimal cell is a mammalian cell, e.g., from a primate, ape, equine,bovine, porcine, canine, feline or rodent. In some cases, a rodentincludes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, orguinea pig.

Exemplary mammalian host cells include, but are not limited to, 293Acell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44 cells,CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F™ cells, F1p-In™T-REx™ 293 cell line, F1p-In™-293 cell line, F1p-In™-3T3 cell line,F1p-In™-BHK cell line, F1p-In™-CHO cell line, F1p-In™-CV-1 cell line,F1p-In™-Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-Scells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells,T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHOcell line, and T-REx™-HeLa cell line.

In some instances, a mammalian host cell is a stable cell line, or acell line that has incorporated a genetic material of interest into itsown genome and has the capability to express the product of the geneticmaterial after many generations of cell division. In some cases, amammalian host cell is a transient cell line, or a cell line that hasnot incorporated a genetic material of interest into its own genome anddoes not have the capability to express the product of the geneticmaterial after many generations of cell division.

Exemplary insect host cells include, but are not limited to, DrosophilaS2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

In some instances, plant cells include a cell from algae. Exemplaryinsect cell lines include, but are not limited to, strains fromChlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a bispecific antibody comprising a firstantigen-binding site that specifically binds to CD3 and a secondantigen-binding site that specifically binds to a tumor antigen.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises the bispecific antibody of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Methods of Treatment

In some embodiments, the isolated polypeptide comprising the cleavablelinkers described herein are used in a method of treating cancer. Insome embodiments, the cancer has cells that express EGFR. In someembodiments, the polypeptides or polypeptide complexes described hereinare used in a method of treating colorectal cancer (CRC), squamous cellcarcinoma of the head and Neck (SCCHN), non-small cell lung cancer(NSCLC), prostate cancer, breast cancer, colon/rectum cancer, head andneck cancer, esophagogastric cancer, liver cancer, glioblastoma,cervical cancer, ovarian cancer, bladder cancer, kidney cancer, orpancreatic cancer. In some embodiments, the polypeptides or polypeptidecomplexes described herein are used in a method of treating subjects whoare resistant to EGFR inhibitor treatment. In some embodiments, thepolypeptides or polypeptide complexes described herein are used in amethod of treating subjects who harbor KRAS mutations. In someembodiments, the polypeptides or polypeptide complexes described hereinare used in a method of treating subjects who are resistant to EGFRinhibitor treatment and harbor KRAS mutations.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EMBODIMENTS

Embodiment 1 comprises an isolated polypeptide comprising a cleavablelinker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG).

Embodiment 2 comprises an isolated polypeptide of embodiment 1, whereinthe cleavable linker comprises the amino acid sequence of SEQ ID NO: 3(ISSGLLSGRSDAG).

Embodiment 3 comprises an isolated polypeptide of any one of embodiments1-2, wherein the cleavable linker comprises the amino acid sequence ofSEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).

Embodiment 4 comprises an isolated polypeptide of any one of embodiments1-3, wherein the cleavable linker comprises the amino acid sequence ofSEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

Embodiment 5 comprises an isolated polypeptide of any one of embodiments1-4, wherein the cleavable linker comprises the amino acid sequence ofSEQ ID NO: 5 (SPLGLSGRSDAG).

Embodiment 6 comprises an isolated polypeptide of any one of embodiments1-5, wherein the cleavable linker comprises the amino acid sequence ofSEQ ID NO: 6 (LSGRSDAGSPLGLAG).

Embodiment 7 comprises an isolated polypeptide of any one of embodiments1-6, wherein the cleavable linker is cleavable by a protease.

Embodiment 8 comprises an isolated polypeptide of embodiment 7, whereinthe protease comprises a tumor specific protease.

Embodiment 9 comprises an isolated polypeptide of any one of embodiments7-8, wherein the protease comprises a matrix metalloprotease (MMP) or aserine protease.

Embodiment 10 comprises an isolated polypeptide of embodiment 9, whereinthe matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

Embodiment 11 comprises an isolated polypeptide of embodiment 9, whereinthe serine protease comprises matriptase, urokinase, or hepsin.

Embodiment 12 comprises an isolated polypeptide of any one ofembodiments 1-11, wherein the isolated polypeptide further comprises anantigen binding domain that binds to a target antigen.

Embodiment 13 comprises an isolated polypeptide of embodiment 12,wherein the antigen binding domain is C-terminal to the cleavablelinker.

Embodiment 14 comprises an isolated polypeptide of any one ofembodiments 1-11, wherein the isolated polypeptide further comprises acytokine or cytokine fragment that binds to a cytokine receptor.

Embodiment 15 comprises an isolated polypeptide of embodiment 14,wherein the cytokine or cytokine fragment is C-terminal to the cleavablelinker.

Embodiment 16 comprises an isolated polypeptide of any one ofembodiments 1-15, wherein the cleavable linker connects a peptide to anantigen binding domain that binds to a target antigen or to a cytokineor cytokine fragment that binds to a cytokine receptor in aconfiguration according to Formula I: A₁-L₁-P₁ wherein A₁ comprises theantigen binding domain that binds to the target antigen or the cytokineor cytokine fragment that binds to the cytokine receptor; L₁ comprisesthe cleavable linker; P₁ comprises a peptide that impairs binding of theantigen binding domain to the target antigen or impairs binding of thecytokine to the cytokine receptor.

Embodiment 17 comprises an isolated polypeptide of embodiment 16,wherein P₁ is connected N-terminal to the cleavable linker and A₁ isconnected C-terminal to the cleavable linker.

Embodiment 18 comprises an isolated polypeptide of embodiment 16,wherein P₁ is connected C-terminal to the cleavable linker and A₁ isconnected N-terminal to the cleavable linker.

Embodiment 19 comprises an isolated polypeptide of any one ofembodiments 16-18, wherein P₁ is bound to A₁ through ionic interactions,electrostatic interactions, hydrophobic interactions, Pi-stackinginteractions, and H-bonding interactions, or a combination thereof.

Embodiment 20 comprises an isolated polypeptide of any one ofembodiments 16-19, wherein P₁ has less than 70% sequence homology to thetarget antigen or the cytokine receptor.

Embodiment 21 comprises an isolated polypeptide of any one ofembodiments 16-20, wherein P₁ comprises a peptide sequence of at least10 amino acids in length.

Embodiment 22 comprises an isolated polypeptide of any one ofembodiments 16-21, wherein P₁ comprises a peptide sequence of at least10 amino acids in length and no more than 20 amino acids in length.

Embodiment 23 comprises an isolated polypeptide of any one ofembodiments 16-22, wherein P₁ comprises a peptide sequence of at least16 amino acids in length.

Embodiment 24 comprises an isolated polypeptide of any one ofembodiments 16-23, wherein P₁ comprises a peptide sequence of no morethan 40 amino acids in length.

Embodiment 25 comprises an isolated polypeptide of any one ofembodiments 16-24, wherein P₁ comprises a cyclic peptide or a linearpeptide.

Embodiment 26 comprises an isolated polypeptide of any one ofembodiments 16-25, wherein P₁ comprises a cyclic peptide.

Embodiment 27 comprises an isolated polypeptide of any one ofembodiments 16-26, wherein P₁ is further linked to a half-life extendingmoiety.

Embodiment 28 comprises an isolated polypeptide of embodiment 27,wherein the half-life extending moiety is a single-domain antibody.

Embodiment 29 comprises an isolated polypeptide of embodiment 28,wherein the single domain antibody comprises 10G.

Embodiment 30 comprises an isolated polypeptide of any one ofembodiments 16-29, wherein A₁ comprises an antibody, a single chainvariable fragment (scFv), a heavy chain variable domain (VH domain), alight chain variable domain (VL domain), a variable domain (VHH) of acamelid derived single domain antibody, a Fab, a Fab′, a Fab light chainpolypeptide, or a Fab heavy chain polypeptide.

Embodiment 31 comprises an isolated polypeptide of any one ofembodiments 16-30, wherein the target antigen comprises a tumor antigen.

Embodiment 32 comprises an isolated polypeptide of any one ofembodiments 30-31, wherein A₁ comprises the Fab light chain polypeptideor the Fab heavy chain polypeptide.

Embodiment 33 comprises an isolated polypeptide of any one ofembodiments 16-32, wherein A₁ comprises an epidermal growth factorreceptor (EGFR) binding domain.

Embodiment 34 comprises an isolated polypeptide of any one ofembodiments 16-30, wherein the target antigen comprises an effector cellantigen.

Embodiment 35 comprises an isolated polypeptide of embodiment 34,wherein A₁ comprises the scFv.

Embodiment 36 comprises an isolated polypeptide of embodiment 35,wherein the scFv comprises an anti-CD3e single chain variable fragment.

Embodiment 37 comprises an isolated polypeptide of any one ofembodiments 16-29, wherein A₁ comprises the cytokine.

Embodiment 38 comprises an isolated polypeptide of embodiment 37,wherein the cytokine or cytokine fragment is a wild-type cytokine.

Embodiment 39 comprises an isolated polypeptide of embodiment 37,wherein the cytokine or cytokine fragment is a mutein of the cytokine.

Embodiment 40 comprises an isolated polypeptide of any one ofembodiments 37-39, wherein the cytokine receptor is an interferonreceptor or an interleukin receptor.

Embodiment 41 comprises an isolated polypeptide of any one ofembodiments 37-40, wherein the cytokine receptor comprises an interferonreceptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6 receptor,IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-21receptor, or TGF-β receptor.

Embodiment 42 comprises an isolated polypeptide of any one ofembodiments 37-41, wherein the cytokine or cytokine fragment comprisesan interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

Embodiment 43 comprises an isolated polypeptide of any one ofembodiments 37-42, wherein the cytokine or cytokine fragment comprisesan IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

Embodiment 44 comprises an isolated polypeptide of any one ofembodiments 1-43, wherein the isolated polypeptide is complexed with asecond isolated polypeptide comprising a second antigen binding domainor a second cytokine or second cytokine fragment.

Embodiment 45 comprises an isolated polypeptide of embodiment 44,wherein the second isolated polypeptide is in a configuration accordingto Formula II: A₂-L₂-P₂ wherein A₂ comprises the second antigen bindingdomain or the second cytokine; L₂ comprises a second cleavable linker;P₂ comprises a second peptide that impairs binding of the second antigenbinding domain to a second target antigen or impairs binding of thesecond cytokine or second cytokine fragment to a second cytokinereceptor.

Embodiment 46 comprises an isolated polypeptide of embodiment 45,wherein the second cleavable linker comprises the amino acid sequence ofSEQ ID NO: 1 (LSGRSDAG).

Embodiment 47 comprises an isolated polypeptide of any one ofembodiments 45-46, wherein the second cleavable linker comprises theamino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG).

Embodiment 48 comprises an isolated polypeptide of any one ofembodiments 45-47, wherein the second cleavable linker comprises theamino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).

Embodiment 49 comprises an isolated polypeptide of any one ofembodiments 45-48, wherein the second cleavable linker comprises theamino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

Embodiment 50 comprises an isolated polypeptide of any one ofembodiments 45-49, wherein the second cleavable linker comprises theamino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG).

Embodiment 51 comprises an isolated polypeptide of any one ofembodiments 45-50, wherein the second cleavable linker comprises theamino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

Embodiment 52 comprises an isolated polypeptide of any one ofembodiments 45-51, wherein P₂ is connected N-terminal to the secondcleavable linker and A₂ is connected C-terminal to the second cleavablelinker.

Embodiment 53 comprises an isolated polypeptide of any one ofembodiments 45-51, wherein P₂ is connected C-terminal to the secondcleavable linker and A₂ is connected N-terminal to the second cleavablelinker.

Embodiment 54 comprises an isolated polypeptide of any one ofembodiments 45-53, wherein P₂ is bound to A₂ through ionic interactions,electrostatic interactions, hydrophobic interactions, P₁-stackinginteractions, and H-bonding interactions, or a combination thereof.

Embodiment 55 comprises an isolated polypeptide of any one ofembodiments 45-54, wherein P₂ has less than 70% sequence homology to thesecond target antigen or the second cytokine receptor.

Embodiment 56 comprises an isolated polypeptide of any one ofembodiments 45-55, wherein P₂ comprises a peptide sequence of at least10 amino acids in length.

Embodiment 57 comprises an isolated polypeptide of any one ofembodiments 45-56, wherein P₂ comprises a peptide sequence of at least10 amino acids in length and no more than 20 amino acids in length.

Embodiment 58 comprises an isolated polypeptide of any one ofembodiments 45-57, wherein P₂ comprises a peptide sequence of at least16 amino acids in length.

Embodiment 59 comprises an isolated polypeptide of any one ofembodiments 45-56, wherein P₂ comprises a peptide sequence of no morethan 40 amino acids in length.

Embodiment 60 comprises an isolated polypeptide of any one ofembodiments 45-59, wherein P₂ comprises a cyclic peptide or a linearpeptide.

Embodiment 61 comprises an isolated polypeptide of any one ofembodiments 45-60, wherein P₂ comprises a cyclic peptide.

Embodiment 62 comprises an isolated polypeptide of any one ofembodiments 45-61, wherein A₂ comprises an antibody, a single chainvariable fragment (scFv), a heavy chain variable domain (VH domain), alight chain variable domain (VL domain), a variable domain (VHH) of acamelid derived single domain antibody, a Fab, a Fab′, a Fab light chainpolypeptide, or a Fab heavy chain polypeptide.

Embodiment 63 comprises an isolated polypeptide of any one ofembodiments 45-62, wherein the second target antigen comprises a tumorantigen.

Embodiment 64 comprises an isolated polypeptide of embodiment 62,wherein A₂ comprises the Fab light chain polypeptide or the Fab heavychain polypeptide.

Embodiment 65 comprises an isolated polypeptide of any one ofembodiments 45-64, wherein A₂ comprises an epidermal growth factorreceptor (EGFR) binding domain.

Embodiment 66 comprises an isolated polypeptide of any one ofembodiments 45-62, wherein the second target antigen comprises aneffector cell antigen.

Embodiment 67 comprises an isolated polypeptide of embodiment 62,wherein A₂ comprises the scFv.

Embodiment 68 comprises an isolated polypeptide of any one ofembodiments 66-67, wherein the scFv comprises an anti-CD3e single chainvariable fragment.

Embodiment 69 comprises an isolated polypeptide of any one ofembodiments 45-61, wherein A₂ comprises the second cytokine.

Embodiment 70 comprises an isolated polypeptide of embodiment 69,wherein the second cytokine or second cytokine fragment is a wild-typecytokine.

Embodiment 71 comprises an isolated polypeptide of embodiment 69,wherein the second cytokine or second cytokine fragment is a mutein ofthe cytokine.

Embodiment 72 comprises an isolated polypeptide of any one ofembodiments 69-71, wherein the second cytokine receptor is an interferonreceptor or an interleukin receptor.

Embodiment 73 comprises an isolated polypeptide of any one ofembodiments 69-72, wherein the second cytokine receptor comprises aninterferon receptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor,IL-21 receptor, or TGF-β receptor.

Embodiment 74 comprises an isolated polypeptide of any one ofembodiments 69-73, wherein the second cytokine or second cytokinefragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, orIL-21.

Embodiment 75 comprises an isolated polypeptide of any one ofembodiments 69-74, wherein the second cytokine or second cytokinefragment comprises an IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

Embodiment 76 comprises a pharmaceutical composition comprising: theisolated polypeptide comprising a cleavable linker of any one of theabove embodiments; and a pharmaceutically acceptable excipient.

Embodiment 77 comprises an isolated recombinant nucleic acid moleculeencoding the isolated polypeptide comprising a cleavable linker of anyone of the above embodiments.

Embodiment 78 comprises a vector comprising the isolated recombinantnucleic acid molecule according to Embodiment 77.

Embodiment 79 comprises a method of producing an isolated polypeptidecomprising a cleavable linker according to any of the above embodimentscomprising culturing a cell under conditions that lead to expression ofthe polypeptide, wherein the cell comprises the vector of embodiment 78.

Embodiment 80 comprises a method of manufacturing an isolatedpolypeptide comprising a cleavable linker, the method comprising: (a)culturing a cell comprising the recombinant nucleic acid molecule ofembodiment 77 under conditions that lead to expression of thepolypeptide, and (b) isolating the polypeptide.

Examples Example 1. Proteolysis Rates and Serum Stability

The polypeptide complexes were evaluated for tumor and serum proteaseactivity.

Briefly, polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 weregenerated comprising peptide masks genetically fused to the polypeptidecomplexes using cleavable linkers recognized by various tumor proteases.The polypeptide complexes were exposed to various tumor proteases.Cleavage rate was determined when the polypeptide complexes were exposedto MMP2, MMP7, MMP9, MMP13, MMP14, uPa, MTSP1, and Hepsin. The data forapparent cleavage rate and relative serum stability are seen in Tables2-4.

TABLE 2 Apparent Cleavage Rate Comparisons Tumor Proteases  PC-1 PC-2PC-3 PC-4 PC-5 MMP2  45  45  5  5  5  10 < r < 10  10 < r <10 >10 >10 >10 MMP7  <2.5 × 10  <2.5 × 10³  45  5  5  10 < r <10 >10 >10 MMP9  <2.5 ×10  <2.5 × 10³  45  5  5  10 <r<10 >10 >10 MMP13 45  45  5  5  5  10 < r < 10  10 < r < 10 >10 >10 >10 MMP14  45  45  45 45  45  10 < r < 10  10 < r < 10  10 < r < 10  10 < r < 10  10 < r < 10uPa  2.5 × 10³ < r < 10⁴  2.5 × 10³ < r < 10  2.5 × 10³ < r < 10⁴  2.5 ×10³ < r < 10⁴  2.5 × 10³ < r < 10⁴ MTSP1  2.5 × 10³ < r < 10⁴  2.5 × 10³< r < 10⁴  45  45  45  10 < r < 10  10 < r < 10  10 < r < 10 Hepsin  5 5  45  45  45 >10 >10  10 < r < 10  10 < r <10  10 < r < 10

TABLE 3 Apparent Cleavage Rate Constants Apparent Cleavage RateConstants (M⁻¹ s⁻¹) (EGFR side) Tumor Protease PC-1 PC-2 PC-3 PC-4 PC-5MMP2 1.99E+04 2.47E+04 3.63E+05 3.29E+05 3.48E+05 MMP7 1.50E+02 1.57E+021.34E+04 1.76E+05 9.15E+04 MMP9 2.35E+03 5.00E+01 4.10E+04 2.36E+052.82E+05 MMP13 1.89E+04 1.20E+04 1.56E+05 2.75E+05 3.80E+05 MMP141.01E+04 1.07E+04 2.78E+04 1.42E+04 1.95E+04 uPa 8.39E+03 4.32E+033.41E+03 4.03E+03 5.88E+03 MTSP1 9.96E+03 8.63E+03 3.83E+04 3.92E+045.65E+04 Hepsin 2.53E+05 2.53E+05 7.00E+04 4.26E+04 8.28E+04

TABLE 4 Relative Serum Stability Relative serum stability (% cleavageper day) (EGFR side/CD3e side) Serum PC-1 PC-2 PC-3 PC-4 PC-5 Human0.51%/ 0.52%/ 0.44%/ 0.44%/ 0.72%/ 0.56% 0.65% 0.63% 0.82% 0.71%

The data shows that serum proteolytic activity is greater than blood.The data also shows the cleavable linker sequences have increased ratesof proteolysis while retaining stability in human serum.

Example 2. Confirmation of Comparable Masking with Cleavable Linkers

The polypeptide complexes were evaluated for EGFR and CD36 binding.

Briefly, the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4,and PC-5 comprising EGFR masking was determined. As seen in FIG. 1A,EGFR masking blocks binding for the various polypeptide complexes.Following cleavage by the tumor protease MTSP1, the polypeptidecomplexes are able to bind (FIG. 1B).

Details of the EGFR binding shifts are seen in Tables 5-8.

TABLE 5 Step Time pH Baseline: Octet buffer  60 sec pH 7.4 Load: 30 nMEGFR-biotin 300 sec pH 7.4 (2.169 ug/mL) Biocytin quench (100 uM) 300sec pH 7.4 Baseline: Octet buffer  90 sec pH 7.4 Association: 300 sec pH7.4 25 nM PC-2 25 nM PC-3 25 nM PC-4 25 nM PC-5 25 nM PC-2 + MTSP1 25nMPC-3 + MTSP1 25 nM PC-4 + MTSP1 25 nM PC-5 + MTSP1 Dissociation: Octetbuffer 600 sec pH 7.4

TABLE 6 Loading Conc. KD Sample ID Sample ID (nM) KD (M) Error kon(1/Ms)kdis(1/s) Full R{circumflex over ( )}2 Full X{circumflex over ( )}2Response PC-2 EGFR 25 No significant binding PC-3 EGFR 25 No significantbinding PC-4 EGFR 25 No significant binding PC-5 EGFR 25 No significantbinding PC-2 + MTSP1 EGFR 25 1.34E−09 2.87E−11 3.48E+05 4.66E−04 0.97352.3843 1.5573 PC-3 + MTSP1 EGFR 25 9.46E−10 1.99E−11 4.72E+05 4.46E−040.9684 2.3105 1.5016 PC-4 + MTSP1 EGFR 25 1.19E−09 2.56E−11 3.82E+054.54E−04 0.9715 2.45   1.553  PC-5 + MTSP1 EGFR 25 1.45E−09 2.98E−113.50E+05 5.07E−04 0.9728 1.9973 1.4156

TABLE 7 Step Time pH Baseline: Octet buffer  60 sec pH 7.4 Load: 30 nMEGFR-biotin 300 sec pH 7.4 (2.169 ug/mL) Biocytin quench (100 uM) 300sec pH 7.4 Baseline: 5% human serum 300 sec pH 7.4 Association: 300 secpH 7.4 30 nM PC-1, 30 nM PC-1 + MTSP1 30 nM PC-6, buffer Dissociation:5% human 600 sec pH 7.4 serum

TABLE 8 Loading Conc. KD Sample ID Sample ID (nM) KD (M) Error kon(1/Ms)kdis(1/s) Full R{circumflex over ( )}2 Full X{circumflex over ( )}2Response PC-1 EGFR- 50 No significant binding biotin PC-1 + MTSP1 EGFR-50 1.91E−09 3.85E−11 2.60E+05 4.98E−04 0.965  1.7318 1.2733 biotin PC-6EGFR- 50 1.77E−09 3.19E−11 4.93E+05 8.72E−04 0.9535 1.4956 1.0938 biotinbuffer EGFR- 50 <1.0E−12 6.00E−09 1.29E+04 <1.0E−07 0.5559 0.1511 0.0151biotin

The polypeptide complexes were also evaluated for CD3E binding. Briefly,the binding of the polypeptide complexes PC-1, PC-2, PC-3, PC-4, andPC-5 comprising CD3ε masking was determined. As seen in FIG. 2A, themasks block binding for the various polypeptide complexes. Followingcleavage by the tumor protease MTSP1, the polypeptide complexes are ableto bind (FIG. 2B).

Details of the CD3ε binding shifts are seen in Tables 9-12.

TABLE 9 Step Time pH Baseline: Octet buffer  60 sec pH 7.4 Load: 30 nMCD3e-biotin 300 sec pH 7.4 (0.465 ug/mL) Biocytin quench (100 uM) 300sec pH 7.4 Baseline: Octet buffer  90 sec pH 7.4 Association: 300 sec pH7.4 25 nM PC-2 25 nM PC-3 25 nM PC-4 25 nM PC-5 25 nM PC-2 + MTSP1 25 nMPC-3 + MTSP1 25 nM PC-4 + MTSP1 25 nM PC-5 + MTSP1 Dissociation: Octetbuffer 600 sec pH 7.4

TABLE 10 Loading Conc. Sample ID Sample ID (nM) KD (M) KD Errorkon(1/Ms) kdis(1/s) Full R{circumflex over ( )}2 Full X{circumflex over( )}2 Response PC-2 CD3e 25 No significant binding PC-3 CD3e 25 Nosignificant binding PC-4 CD3e 25 No significant binding PC-5 CD3e 25 Nosignificant binding PC-2 + MTSP1 CD3e 25 1.41E−08 3.66E−10 2.37E+053.33E−03 0.9829 1.2588 1.0342 PC-3 + MTSP1 CD3e 25 1.00E−08 2.60E−103.37E+05 3.37E−03 0.976  2.078  1.1036 PC-4 + MTSP1 CD3e 25 1.19E−083.14E−10 2.78E+05 3.30E−03 0.9786 1.5272 1.0227 PC-5 + MTSP1 CD3e 251.32E−08 3.33E−10 2.91E+05 3.84E−03 0.9832 1.452  1.0526

TABLE 11 Step Time pH Baseline: Octet buffer  60 sec pH 7.4 Load: 30nMCD3e-biotin 300 sec pH 7.4 (0.46ug/mL) Biocytin quench (IOOuM) 300 secpH 7.4 Baseline: 5% human serum 300 sec pH 7.4 Association: 300 sec pH7.4 30nMPC-l, 30nM PC-1 + MTSP1 30nM PC-6, buffer Dissociation: 5% humanserum 600 sec pH 7.4

TABLE 12 Loading Conc. KD Sample ID Sample ID (nM) KD (M) Errorkon(1/Ms) kdis(1/s) Full R{circumflex over ( )}2 Full X{circumflex over( )}2 Response PC-1 CD3e- 50 No significant binding biotin PC-1 + MTSP1CD3e- 50 3.15E−08 1.01E−09 1.25E+05 3.95E−03 0.9774 2.6058 1.2556 biotinPC-6 CD3e- 50 2.26E−08 6.81E−10 1.33E+05 3.02E−03 0.9702 4.4245 1.5652biotin buffer CD3e- 50 No significant binding biotin

The polypeptide complexes binding was evaluated using enzyme linkedimmunosorbent assays (ELISAs). Biotinylated peptides were captured onneutravidin coated plates. A secondary antibody was used to detect boundpolypeptide complex. Data for PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6comprising the mask and following cleavage of the mask is seen in FIGS.3A-3B and EC50 binding data is summarized in Tables 13-14.

TABLE 13 EC50 nM Masked Cleaved Shift PC-2 21.29 0.13 169.5 PC-3 23.470.12 198.2 PC-4 20.59 0.15 139.3 PC-5 18.28 0.13 143.5 PC-1 44.79 —282.8 PC-6 — 0.16 —

TABLE 14 EC50 nM Masked Cleaved Shift PC-2 2.082 0.101 20.6x PC-3 1.7920.1007 17.8x PC-4 2.439 0.119 20.5x PC-5 2.665 0.121 22.0x PC-1 4.5980.1383 33.2x PC-6 — 0.069 —

Example 3. Confirmation of Comparable T-Cell Shifts with CleavableLinkers

The polypeptide complexes were next evaluated in functional in vitrotumor cell killing and related T cell activation studies.

Briefly, HCT116 cells were seeded onto 96 well tissue culture treatedflat bottom plates and allowed to adhere overnight. The following day,culture medium and nonadherent cells were removed and replaced withfresh medium containing titrated the polypeptide complexes atconcentrations indicated. The data for PC-2, PC-3, PC-4, PC-5, PC-1, andPC-6 is seen in FIGS. 4A-4E and Tables 15-16.

TABLE 15 Tumor cell viability, IC50 (pM) Cytotox PC-2 + PC-3 + PC-4 +PC-5 + time PC-6 PC-1 PC-2 PC-3 PC-4 PC-5 MTSP1 MTSP1 MTSP1 MTSP1  24 hr1.13 555.6 326.6 578.3 539.9 869.4 0.2377 0.2811 0.3557 0.2294  48 hr0.4675 460.9 301.8 389.1 419.7 499.6 0.1998 0.1843 0.1596 0.2197  72 hr0.5507 763.5 389.3 444 522.6 588.6 0.2632 0.2402 0.218 0.3065  96 hr0.7548 1023 475.3 511.8 659.1 712.9 0.3296 0.3099 0.2809 0.394 120 hr577.5 589.6 773.2 824.2 0.3901 0.3679 0.3351 0.4737

TABLE 16 Tumor cell viability, Functional Shift Cytotox PC-2 + PC-3 +PC-4 + PC-5 + time PC-6 PC-1 PC-2 PC-3 PC-4 PC-5 MTSP1 MTSP1 MTSP1 MTSP1 24 hr 1x  491.7x   289x 511.8x  477.8x  769.4x 0.2x 0.2x 0.3x 0.2x  48hr 1x  985.9x 645.6x 832.3x  897.8x 1068.7x 0.4x 0.4x 0.3x 0.5x  72 hr1x 1386.4x 706.9x 806.2x    949x 1068.8x 0.5x 0.4x 0.4x 0.6x  96 hr 1x1355.3x 629.7x 678.1x  873.2x  944.5x 0.4x 0.4x 0.4x 0.5x 120 hr 765.1x781.1x 1024.4x 1091.9x 0.5x 0.5x 0.4x 0.6x

Example 4. In Vivo Cynomolgus Monkey PK Comparison

The polypeptide complexes were assessed for pharmacokinetic and safetyin cynomolgus monkey.

Cynomolgus Monkeys

Young naïve male cynomolgus monkeys were paired housed by group andidentified by unique body tattoo. All animals were acclimated to housingconditions for 3 days prior to the start of the study. Prior toinitiation all animals had undergone a physical examination by the studyveterinarian. Only animals that, in the opinion of the studyveterinarian, were healthy and otherwise met the criteria were admittedto the study. Food was withheld overnight prior to dosing. Purina 5049was provided daily in amounts appropriate for the size of the animal.Tap water was provided ad libitum via automatic watering device.

Pharmacokinetics

Polypeptide complex pharmacokinetics for polypeptide complexes PC-1,PC-2, PC-3, PC-4, and PC-5 were determined in naïve male cynomolgusmonkeys weighing 2-3 kg. Briefly, two group housed monkeys were used perdosing group and allowed to acclimate to their surroundings prior todosing. Animals were sedated with Ketamine HCL 10-20 mg/kg IM prior todosing and bleeding. Concentrated test articles were diluted in sterilephosphate buffered saline and administered to animals at a quantityrelative to the animals' mass in kg. The dose for each test article wasadministered intravenously at 1 mL/kg dosing volume. For dosing, theleft and right limbs were clipped and prepped with alcohol. Thesaphenous vein was identified, and a standard catheter was placed for IVbolus infusion (in either the left or right limb). The test articledosing solution was attached to the catheter via syringe and the bolusinfusion occurred via manual compression of the syringe.

For blood collections, animals were sedated using ketamine, the femoraltriangle was prepared, and blood was collected from the femoral veinusing a 22G 1.5 inch needle, vacutainer sheath, and collection tube.Following venipuncture, manual compression of the vein was maintaineduntil hemostasis was achieved. Blood collections were based on weight ofthe animals and did not exceed AGI maximum bleeds as set forth by IACUC.Blood was collected in EDTA tubes and processed to plasma. The bloodsamples were centrifuged cold at 3000×g for 10 min to separate cellsfrom plasma. The plasma supernatant was harvested and stored frozenprior to analysis.

The concentration of the polypeptide complexes in cyno plasma sampleswas determined by ELISA. Briefly, anti-histag capture antibody wascoated directly on ELISA plates. Standard dilutions of polypeptidecomplex in cyno serum were used to generate a calibration curve to whichanimal PK test samples could be compared. Standards and test sampleswere added to the plate and incubated cold overnight. Several differentdilutions of test samples were used to make sure signals landed withinappropriate dynamic range of the standard curve. Plates were washed andincubated with an anti-human HRP detection antibody for a brief time.Plates were washed, developed, and stopped using standard ELISAtechniques. Standard curves plotting absorbance at 450 nm versus knownpolypeptide complex concentration were used to calculate theconcentration of unknown test articles in each mouse PK plasma sample.Concentration of polypeptide complexes were plotted versus time and fitto a standard two stage distribution and elimination pharmacokineticmodel. The calculated pharmacokinetic and parameters for polypeptidecomplexes PC-1, PC-7, PC-4, and PC-5 from cynomolgus monkey are shown inFIGS. 5A-5D and Tables 17-20.

TABLE 17 Dose C_(max) T_(1/2) Vd CL AUC_((0→d7)) Construct (ug/kg) (nM)(hr) (L) (ml/h/kg) (nM · min) PC-1 100 51 100 0.06 0.14 165,895 PC-2 100PC-3 100 PC-4 100 48 91 0.07 0.16 221,875 PC-5 100 61 97 0.05 0.12244,051

TABLE 18 Cyno PK Parameters CL Dose C_(max) T_(1/2) Vd (ml/ Construct(ug/kg) (nM) (hr) (L) h/kg) PC-1 300 153 109 0.06  0.13 PC-1 100  51 1000.06  0.14 PC-7  10  1.7  1.2 0.24  47 PC-7  3  0.17  0.3 0.68 491

TABLE 19 Dose C_(max) T_(1/2) Vd CL Construct (ug/kg) (nM) (hr) (L)(ml/h/kg) PC-1 100 51 100 0.06 0.14

TABLE 20 Parameter PC-2 100 ug/kg PC-3 100 ug/kg Units CMAX 40.37 53.78nM t1/2 69.44 104.57 hr Vd 0.08 0.06 L VSS 0.06 0.11 L CL 0.26 0.13mL/hr/kg BW 3.00 3.00 kg 7 day AUC 207834 305952 nM · min

Example 5. In Vivo Cynomolgus Monkey Cytokine Release

Cytokine release was measured in cynomolgus monkeys.

Cytokines present in plasma post treatment were measured using thenon-human primate Th1/Th2 cytometric bead array assay kit from BDBiosciences (Cat no. 557800) according to the manufacturer'sinstructions. Data is shown in FIGS. 6A-61 ) and Table 21.

TABLE 21 Property PC-7 PC-7 PC-1 PC-2 PC-3 PC-4 PC-5 3 ug/kg 10 ug/kg100 ug/kg Plasma Cytokine Levels (Cmax pg/ml) IL-6 1,932 5,352 BQL TBDTBD 234 493 TNFα 1,267 2,497 BQL TBD TBD BQL BQL IFNγ BQL 109 BQL TBDTBD BQL BQL IL-2 87 412 BQL TBD TBD BQL BQL

Example 6. In Vivo Cynomolgus Monkey Liver ALT/AST

ALT/AST levels were measured. As seen in FIGS. 7A and 7B and Table 22,polypeptide complexes prevented liver toxicity in cynomolgus monkeys.

TABLE 22 PC-1 PC-2 PC-3 PC-4 PC-5 Property 100 ug/kg Plasma Levels (U/L)ALT 18 TBD TBD 40 41 AST  9 TBD TBD 40 51

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-30. (canceled)
 31. An isolated polypeptide comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).
 32. The isolated polypeptide of claim 31, wherein the cleavable linker is cleavable by a protease.
 33. The isolated polypeptide of claim 32, wherein the protease comprises a tumor specific protease.
 34. The isolated polypeptide of claim 32, wherein the protease comprises a matrix metalloprotease (MMP) or a serine protease.
 35. The isolated polypeptide of claim 34, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
 36. The isolated polypeptide of claim 34, wherein the serine protease comprises matriptase, urokinase, or hepsin.
 37. The isolated polypeptide of claim 31, wherein the isolated polypeptide further comprises an antigen binding domain that binds to a target antigen.
 38. The isolated polypeptide of claim 37, wherein the cleavable linker connects a peptide to the antigen binding domain that binds to the target antigen in a configuration according to Formula I: A₁-L₁-P₁ wherein A₁ comprises the antigen binding domain that binds to the target antigen; L₁ comprises the cleavable linker; P₁ comprises a peptide that impairs binding of the antigen binding domain to the target antigen; and wherein P₁ is connected C-terminal to L₁ and A₁ is connected N-terminal to L₁, or P₁ is connected N-terminal to L₁ and A₁ is connected C-terminal to L₁.
 39. The isolated polypeptide of claim 38, wherein P₁ is further linked to a half-life extending moiety, and wherein the half-life extending moiety is a single-domain antibody.
 40. The isolated polypeptide of claim 38, wherein A₁ comprises an antibody, a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain (VHH) of a camelid derived single domain antibody, a Fab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chain polypeptide.
 41. The isolated polypeptide of claim 40, wherein A₁ comprises the scFv.
 42. The isolated polypeptide of claim 41, wherein the scFv comprises an anti-CD3e single chain variable fragment.
 43. A complex comprising the isolated polypeptide of claim 37 and a second isolated polypeptide comprising a second antigen binding domain.
 44. The isolated polypeptide of claim 43, wherein the second isolated polypeptide is in a configuration according to Formula II: A₂-L₂-P₂ wherein A₂ comprises the second antigen binding domain; L₂ comprises a second cleavable linker; P₂ comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen; wherein the second antigen binding domain comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide and the second target antigen comprises a tumor antigen.
 45. The isolated polypeptide of claim 44, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG).
 46. The isolated polypeptide of claim 44, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG).
 47. The isolated polypeptide of claim 44, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).
 48. The isolated polypeptide of claim 44, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).
 49. The isolated polypeptide of claim 44, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG).
 50. The isolated polypeptide of claim 44, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 